Glycosylated interferon alpha

ABSTRACT

The invention relates to interferon-α molecules having certain O-linked oligosaccharide structures.

RELATED APPLICATION INFORMATION

This application is a continuation of U.S. patent application Ser. No.11/973,853, filed Oct. 10, 2007, the disclosure of which is incorporatedin its entirety herein by reference, which claims the benefit of U.S.provisional patent application Nos. 60/857,896, filed Nov. 9, 2006;60/877,601, filed Dec. 28, 2006; and 60/918,504, filed Mar. 16, 2007,the disclosures of which are incorporated in their entirety herein byreference, and which is a continuation-in-part of U.S. patentapplication Ser. No. 11/708,598, filed Feb. 20, 2007, now U.S. Pat. No.7,511,120, issued Mar. 31, 2009, the disclosure of which is incorporatedin its entirety herein by reference, which claims the benefit of U.S.provisional patent application Nos. 60/783,648, filed Mar. 17, 2006; and60/840,291, filed Aug. 25, 2006, the disclosures of which areincorporated in their entirety herein by reference, and which is acontinuation-in-part of U.S. patent application Ser. No. 11/370,555,filed Mar. 8, 2006, now issued U.S. Pat. No. 7,338,654, issued Mar. 4,2008, the disclosure of which is incorporated in its entirety herein byreference, which is a continuation of U.S. patent application Ser. No.10/351,196, filed Jan. 24, 2003, now U.S. Pat. No. 7,129,390, issuedOct. 31, 2006, the disclosure of which is incorporated in its entiretyherein by reference.

BACKGROUND

Numerous natural and synthetic proteins are used in diagnostic andtherapeutic applications; many others are in development or in clinicaltrials. Current methods of protein production include isolation fromnatural sources and recombinant production in bacterial and mammaliancells. Because of the complexity and high cost of these methods ofprotein production, however, efforts are underway to developalternatives. For example, methods for producing exogenous proteins inthe milk of pigs, sheep, goats, and cows have been reported. Theseapproaches have certain limitations, including long generation timesbetween founder and production transgenic herds, extensive husbandry andveterinary costs, and variable levels of expression because of positioneffects at the site of the transgene insertion in the genome. Proteinsare also being produced using milling and malting processes from barleyand rye. However, plant post-translational modifications differ fromvertebrate post-translational modifications, which often has a criticaleffect on the function of the exogenous proteins such as pharmaceuticalproteins.

Like tissue culture and mammary gland bioreactors, the avian oviduct canalso potentially serve as a bioreactor. Successful methods of modifyingavian genetic material such that high levels of exogenous proteins aresecreted in the oviduct and packaged into eggs would allow inexpensiveproduction of large amounts of protein. Several advantages of such anapproach would be: a) short generation times (24 weeks) and rapidestablishment of transgenic flocks via artificial insemination; b)readily scaled production by increasing flock sizes to meet productionneeds; c) post-translational modification of expressed proteins; 4)automated feeding and egg collection; d) naturally sterile egg-whites;and e) reduced processing costs due to the high concentration of proteinin the egg white.

The avian reproductive system, including that of the chicken, is welldescribed. The egg of the hen consists of several layers which aresecreted upon the yolk during its passage through the oviduct. Theproduction of an egg begins with formation of the large yolk in theovary of the hen. The unfertilized oocyte is then positioned on top ofthe yolk sac. Upon ovulation or release of the yolk from the ovary, theoocyte passes into the infundibulum of the oviduct where it isfertilized if sperm are present. It then moves into the magnum of theoviduct which is lined with tubular gland cells. These cells secrete theegg-white proteins, including ovalbumin, lysozyme, ovomucoid,conalbumin, and ovomucin, into the lumen of the magnum where they aredeposited onto the avian embryo and yolk.

The ovalbumin gene encodes a 45 kD protein that is specificallyexpressed in the tubular gland cells of the magnum of the oviduct (BeatoCell 56:335-344 (1989)). Ovalbumin is the most abundant egg whiteprotein, comprising over 50 percent of the total protein produced by thetubular gland cells, or about 4 grams of protein per large Grade A egg(Gilbert, “Egg albumen and its formation” in Physiology and Biochemistryof the Domestic Fowl, Bell and Freeman, eds., Academic Press, London,N.Y., pp. 1291-1329). The ovalbumin gene and over 20 kb of each flankingregion have been cloned and analyzed (Lai et al., Proc. Natl. Acad. Sci.USA 75:2205-2209 (1978); Gannon et al., Nature 278:428-424 (1979); Roopet al., Cell 19:63-68 (1980); and Royal et al., Nature 279:125-132(1975)).

Much attention has been paid to the regulation of the ovalbumin gene.The gene responds to steroid hormones such as estrogen, glucocorticoids,and progesterone, which induce the accumulation of about 70,000ovalbumin mRNA transcripts per tubular gland cell in immature chicks and100,000 ovalbumin mRNA transcripts per tubular gland cell in the maturelaying hen (Palmiter, J. Biol. Chem. 248:8260-8270 (1973); Palmiter,Cell 4:189-197 (1975)). DNAse hypersensitivity analysis andpromoter-reporter gene assays in transfected tubular gland cells defineda 7.4 kb region as containing sequences required for ovalbumin geneexpression. This 5′ flanking region contains four DNAse I-hypersensitivesites centered at −0.25, −0.8, −3.2, and −6.0 kb from the transcriptionstart site. These sites are called HS-I, -II, -III, and -IV,respectively. These regions reflect alterations in the chromatinstructure and are specifically correlated with ovalbumin gene expressionin oviduct cells (Kaye et al., EMBO 3:1137-1144 (1984)).Hypersensitivity of HS-II and -III are estrogen-induced, supporting arole for these regions in hormone-induction of ovalbumin geneexpression.

HS-I and HS-II are both required for steroid induction of ovalbumin genetranscription, and a 1.4 kb portion of the 5′ region that includes theseelements is sufficient to drive steroid-dependent ovalbumin expressionin explanted tubular gland cells (Sanders and McKnight, Biochemistry 27:6550-6557 (1988)). HS-I is termed the negative-response element (“NRE”)because it contains several negative regulatory elements which repressovalbumin expression in the absence of hormones (Haekers et al., Mol.Endo. 9:1113-1126 (1995)). Protein factors bind these elements,including some factors only found in oviduct nuclei suggesting a role intissue-specific expression. HS-II is termed the steroid-dependentresponse element (“SDRE”) because it is required to promote steroidinduction of transcription. It binds a protein or protein complex knownas Chirp-I. Chirp-I is induced by estrogen and turns over rapidly in thepresence of cyclohexamide (Dean et al., Mol. Cell. Biol. 16:2015-2024(1996)). Experiments using an explanted tubular gland cell culturesystem defined an additional set of factors that bind SDRE in asteroid-dependent manner, including an NFκB-like factor (Nordstrom etal., J. Biol. Chem. 268:13193-13202 (1993); Schweers and Sanders, J.Biol. Chem. 266: 10490-10497 (1991)).

Less is known about the function of HS-III and -IV. HS-III contains afunctional estrogen response element, and confers estrogen inducibilityto either the ovalbumin proximal promoter or a heterologous promoterwhen co-transfected into HeLa cells with an estrogen receptor cDNA.These data imply that HS-III may play a functional role in the overallregulation of the ovalbumin gene. Little is known about the function ofHS-IV, except that it does not contain a functional estrogen-responseelement (Kato et al., Cell 68: 731-742 (1992)).

There has been much interest in modifying eukaryotic genomes byintroducing foreign genetic material and/or by disrupting specificgenes. Certain eukaryotic cells may prove to be superior hosts for theproduction of exogenous eukaryotic proteins. The introduction of genesencoding certain proteins also allows for the creation of new phenotypeswhich could have increased economic value. In addition, somegenetically-caused disease states may be cured by the introduction of aforeign gene that allows the genetically defective cells to express theprotein that they can otherwise not produce. Finally, modification ofanimal genomes by insertion or removal of genetic material permits basicstudies of gene function, and ultimately may permit the introduction ofgenes that could be used to cure disease states, or result in improvedanimal phenotypes.

Transgenesis has been accomplished in mammals by several differentmethods. First, in mammals including the mouse, pig, goat, sheep andcow, a transgene is microinjected into the pronucleus of a fertilizedegg, which is then placed in the uterus of a foster mother where itgives rise to a founder animal carrying the transgene in its germline.The transgene is engineered to carry a promoter with specific regulatorysequences directing the expression of the foreign protein to aparticular cell type. Since the transgene inserts randomly into thegenome, position effects at the site of the transgene's insertion intothe genome may variably cause decreased levels of transgene expression.This approach also requires characterization of the promoter such thatsequences necessary to direct expression of the transgene in the desiredcell type are defined and included in the transgene vector (Hogan et al.Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY(1988)).

A second method for effecting animal transgenesis is targeted genedisruption, in which a targeting vector containing sequences of thetarget gene flanking a selectable marker gene is introduced intoembryonic stem (“ES”) cells. By homologous recombination, the targetingvector replaces the target gene sequences at the chromosomal locus orinserts into interior sequences preventing expression of the target geneproduct. Clones of ES cells having the appropriately disrupted gene areselected and then injected into early stage blastocysts generatingchimeric founder animals, some of which have the transgene in the germline. In the case where the transgene deletes the target locus, itreplaces the target locus with foreign DNA borne in the transgenevector, which consists of DNA encoding a selectable marker useful fordetecting transfected ES cells in culture and may additionally containDNA sequences encoding a foreign protein which is then inserted in placeof the deleted gene such that the target gene promoter drives expressionof the foreign gene (U.S. Pat. Nos. 5,464,764 and 5,487,992 (M. P.Capecchi and K. R. Thomas)). This approach suffers from the limitationthat ES cells are unavailable in many mammals, including goats, cows,sheep and pigs. Furthermore, this method is not useful when the deletedgene is required for survival or proper development of the organism orcell type.

Recent developments in avian transgenesis have allowed the modificationof avian genomes. Germ-line transgenic chickens may be produced byinjecting replication-defective retrovirus into the subgerminal cavityof chick blastoderms in freshly laid eggs (U.S. Pat. No. 5,162,215;Bosselman et al., Science 243:533-534 (1989); Thoraval et al.,Transgenic Research 4:369-36 (1995)). The retroviral nucleic acidcarrying a foreign gene randomly inserts into a chromosome of theembryonic cells, generating transgenic animals, some of which have thetransgene in their germ line. Use of insulator elements inserted at the5′ or 3′ region of the fused gene construct to overcome position effectsat the site of insertion has been described (Chim et al., Cell74:504-514 (1993)).

In another approach, a transgene has been microinjected into thegerminal disc of a fertilized egg to produce a stable transgenic founderavian that may pass the gene to the F1 generation (Love et al.,Bio/Technology 12:60-63 (1994)). However, this method has severaldisadvantages. Hens must be sacrificed in order to collect thefertilized egg, the fraction of transgenic founders is low, and injectedeggs require labor intensive in vitro culture in surrogate shells.

In another approach, blastodermal cells containing presumptiveprimordial germ cells (“PGCs”) are excised from donor eggs, transfectedwith a transgene and introduced into the subgerminal cavity of recipientembryos. The transfected donor cells are incorporated into the recipientembryos generating transgenic embryos, some of which are expected tohave the transgene in the germ line. The transgene inserts in randomchromosomal sites by nonhomologous recombination. However, no transgenicfounder avians have yet been generated by this method.

Lui, Poult. Sci. 68:999-1010 (1995), used a targeting vector containingflanking DNA sequences of the vitellogenin gene to delete part of theresident gene in chicken blastodermal cells in culture. However, it hasnot been demonstrated that these cells can contribute to the germ lineand thus produce a transgenic embryo. In addition, this method is notuseful when the deleted gene is required for survival or properdevelopment of the organism or cell type.

Thus, it can be seen that there is a need for a method of introducingforeign DNA, operably linked to a suitable promoter, into the aviangenome such that efficient expression of an exogenous gene can beachieved. Furthermore, there exists a need to create germ-line modifiedtransgenic avians which express exogenous genes in their oviducts andsecrete the expressed exogenous proteins into their eggs.

When interferon was discovered in 1957, it was hailed as a significantantiviral agent. In the late 1970s, interferon became associated withrecombinant gene technology. Today, interferon is a symbol of thecomplexity of the biological processes of cancer and the value ofendurance and persistence in tackling this complexity.

The abnormal genes that cause cancer comprise at least three types:firstly, there are the oncogenes, which, when altered, encourage theabnormal growth and division that characterize cancer. Secondly, thereare the tumor suppressor genes, which, when altered, fail to controlthis abnormal growth and division. Thirdly, there are the DNA repairgenes, which, when altered, fail to repair mutations that can lead tocancer. Researchers speculate that there are about 30 to 40 tumorsuppressor genes in the body, each of which produces a protein. Theseproteins may be controlled by “master” tumor suppressor proteins such asRb (for retinoblastoma, with which it was first associated) and p53(associated with many different tumors). Evidence from the laboratorysuggests that returning just one of these tumor suppressor genes to itsnormal function can appreciably reduce the aggressiveness of themalignancy.

Scientists became intrigued by interferon when it was discovered thatinterferon can inhibit cell growth. Further, interferon was found tohave certain positive effects on the immune system. It is now consideredanalogous to a tumor suppressor protein: it inhibits the growth ofcells, particularly malignant cells; it blocks the effects of manyoncogenes and growth factors; and unlike other biological agents, itinhibits cell motility which is critical to the process of metastasis.

Intercellular communication is dependent on the proper functioning ofall the structural components of the tissue through which the messagesare conveyed: the matrix, the cell membrane, the cytoskeleton, and thecell itself. In cancer, the communication network between cells isdisrupted. If the cytoskeleton is disrupted, the messages don't getthrough to the nucleus and the nucleus begins to function abnormally.Since the nucleus is the site where the oncogenes or tumor suppressorgenes get switched on or off, this abnormal functioning can lead tomalignancy. When this happens, the cells start growing irregularly anddo not differentiate. They may also start to move and disrupt othercells. It is believed that interferon, probably in concert with otherextracellular and cellular substances, restores the balance andhomeostasis, making sure the messages get through properly. Interferonstops growth, stops motility, and enhances the ability of the cell,through adhesion molecules, to respond to its environment. It alsocorrects defects and injuries in the cytoskeleton. Interferon has beenfound to block angiogenesis, the initial step in the formation of newblood vessels that is essential to the growth of malignancies. Moreover,it blocks fibrosis, a response to injury that stimulates many differentkinds of cells and promotes cell growth (Kathryn L. Hale, Oncolog,Interferon: The Evolution of a Biological Therapy, Taking a New Look atCytokine Biology).

Interferon is produced by animal cells when they are invaded by virusesand is released into the bloodstream or intercellular fluid to inducehealthy cells to manufacture an enzyme that counters the infection. Formany years the supply of human interferon for research was limited bycostly extraction techniques. In 1980, however, the protein becameavailable in greater quantities through genetic engineering (i.e.,recombinant forms of the protein). Scientists also determined that thebody makes three distinct types of interferon, referred to as α-(alpha),β-(beta), and γ-(gamma) interferon. Interferons were first thought to behighly species-specific, but it is now known that individual interferonsmay have different ranges of activity in other species. Alpha interferon(α-IFN) has been approved for therapeutic use against hairy-cellleukemia and hepatitis C. α-IFN has also been found effective againstchronic hepatitis B, a major cause of liver cancer and cirrhosis, aswell as for treatment of genital warts and some rarer cancers of bloodand bone marrow. Nasal sprays containing α-IFN provide some protectionagainst colds caused by rhinoviruses. Human α-IFN belongs to a family ofextra-cellular signaling proteins with antiviral, antiproliferating andimmunomodulatory activities. IFN-α proteins are encoded by a multigenefamily which includes 13 genes clustered on the human chromosome 9. Mostof the IFN-α genes are expressed at the mRNA level in leukocytes inducedby Sendai virus. Further, it has been shown that at least nine differentsub-types are also produced at the protein level. The biologicalsignificance of the expression of several similar IFN-α proteins is notknown, however, it is believed that they have quantitatively distinctpatterns of antiviral, growth inhibitory and killer-cell-stimulatoryactivities. Currently, two IFN-α variants, IFN-α 2a and IFN-α 2b, aremass produced in Escherichia coli by recombinant technology and marketedas drugs.

Unlike natural IFN-α, these recombinant IFN-α products have been shownto be immunogenic in some patients, which could be due to unnaturalforms of IFN-α proteins. Thus, for the development of IFN-α drugs it isnecessary to not only identify the IFN-α subtypes and variants expressedin normal human leukocytes, but also to characterize their possiblepost-translational modifications (Nyman et al. (1998) Eur. J. Biochem.253:485-493).

Nyman et al. (supra) studied the glycosylation of natural human IFN-α.They found that two out of nine of the subtypes produced by leukocytesafter a Sendai-virus induction were found to be glycosylated, namelyIFN-α 14c and IFN-α 2b, which is consistent with earlier studies. IFN-α14 is the only IFN-α subtype with potential N-glycosylation sites, Asn2and Asn72, but only Asn72 is actually glycosylated. IFN-α 2 isO-glycosylated at Threonine 106 (Thr106). Interestingly, no other IFN-αsubtype contains Thr at this position. In this study, Nyman et al.liberated and isolated the oligosaccharide chains and analyzed theirstructures by mass spectrometry and specific glycosidase digestions.Both IFN-α 2b and IFN-α 14c resolved into three peaks in reversed-phasehigh performance liquid chromatography (RP-HPLC). Electrosprayionization mass spectrometry (ESI-MS) analysis of IFN-α 2b fractionsfrom RP-HPLC revealed differences in their molecular masses, suggestingthat these represent different glycoforms. This was confirmed bymasspectrometric analysis of the liberated O-glycans of each fraction.IFN-α 2b was estimated to contain about 20% of the core type-2pentasaccharide, and about 50% of disialylated and 30% of monosialylatedcore type-1 glycans. Nyman et al.'s data agrees with previous partialcharacterization of IFN-α 2b glycosylation (Adolf et al. (1991) Biochem.J. 276:511-518). The role of glycosylation in IFN-α 14c and IFN-α 2b isnot clearly established. According to Nyman et al. (supra), thecarbohydrate chains are not essential for the biological activity, butglycosylation may have an effect on the pharmacokinetics and stabilityof the proteins.

There are at least 15 functional genes in the human genome that code forproteins of the IFN-α family. The amino acid sequence similarities aregenerally in the region of about 90%, thus, these molecules are closelyrelated in structure. IFN-α proteins contain 166 amino acids (with theexception of IFN-α 2, which has 165 amino acids) and characteristicallycontain four conserved cysteine residues which form two disulfidebridges. IFN-α species are slightly acidic in character and lack arecognition site for asparagine-linked glycosylation (with the exceptionof IFN-α 14 which does contain a recognition site for asparagine-linkedglycosylation). Three variants of IFN-α 2, differing in their aminoacids at positions 23 and 34, are known: IFN-α 2a (Lys-23, His-34);IFN-α 2b (Arg-23, His-34); and IFN-α 2c (Arg-23, Arg-34). It is believedthat IFN-α 2a and IFN-α 2c are allelic variants of IFN-α 2b. See, Gewertet al (1993) J. Interferon Res. vol 13, p 227-231. The minor differencesin amino acid content of the IFN-α 2 species is not expected to effectglycosylation of the interferons. That is glycosyation patterns areexpected to be essentially the same for each of IFN-α 2a, 2b and 2c. Twoother human IFN species, namely IFN-ω 1 and IFN-β are N-glycosylated andare more distantly related to IFN-α. IFN-α, -β and -ω, collectivelyreferred to as class I IFNs, bind to the same high affinity cellmembrane receptor (Adolf et al. (1991) Biochem. J. 276:511-518).

Adolf et al. (supra) used the specificity of a monoclonal antibody forthe isolation of natural IFN-α 2 from human leukocyte IFN. They obtaineda 95% pure protein through immunoaffinity chromatography which confirmedthe expected antiviral activity of IFN-α 2. Analysis of natural IFN-α 2by reverse-phase HPLC, showed that the natural protein can be resolvedinto two components, both more hydrophilic than E. coli-derived IFN-α 2.SDS/PAGE revealed that the protein is also heterogeneous in molecularmass, resulting in three bands, all of them with lower electrophoreticmobility than the equivalent E. coli-derived protein.

Adolf et al. (supra) also speculated that natural IFN-α 2 carriesO-linked carbohydrate residues. Their hypothesis was confirmed bycleavage of the putative peptide-carbohydrate bond with alkali; theresulting protein was homogeneous and showed the same molecular mass asthe recombinant protein. Further comparison of natural and recombinantproteins after proteolytic cleavage, followed by separation and analysisof the resulting fragments, allowed them to define a candidateglycopeptide. Sequence analysis of this peptide identified Thr-106 asthe O-glycosylation site. A comparison of the amino acid sequences ofall published IFN-α 2 species revealed that this threonine residue isunique to IFN-α 2. Glycine, isoleucine or glutamic acid are present atthe corresponding position (107) in all other proteins.

Preparations of IFN-α 2 produced in E. coli are devoid ofO-glycosylation and have been registered as drugs in many countries.However, the immunogenicity of therapeutically applied E. coli-derivedIFN-α 2 might be affected by the lack of glycosylation. Studies haveshown that four out of sixteen patients receiving recombinant humangranulocyte-macrophage colony-stimulating factor produced in yeastdeveloped antibodies to this protein. Interestingly, these antibodieswere found to react with epitopes that in the endogenousgranulocyte-macrophage colony-stimulating factor are protected byO-linked glycosylation, but which are exposed in the recombinant factor(Adolf et al., supra).

Similarly, induction of antibodies to recombinant E. coli-derived IFN-α2 after prolonged treatment of patients has been described and it hasbeen speculated that natural IFN-α 2 may be less immunogenic than therecombinant IFN-α 2 proteins (Galton et al. (1989) Lancet 2:572-573).

What is needed are improved methods of producing therapeutic orpharmaceutical proteins such as antibodies and cytokines includinginterferon, G-CSF and erythropoietin.

SUMMARY OF THE INVENTION

This invention provides vectors and methods for the stable introductionof exogenous nucleic acid sequences into the genome of avians in orderto express the exogenous sequences to alter the phenotype of the aviansor to produce desired proteins. In particular, transgenic avians areproduced which express exogenous sequences in their oviducts and whichdeposit exogenous proteins, such as pharmaceutical proteins, into theireggs. Avian eggs that contain such exogenous proteins are encompassed bythis invention. The present invention further provides novel forms oftherapeutic proteins (e.g., human cytokines) including interferons,G-CSF, G-MCSF and erythropoietin which are efficiently expressed in theoviduct of transgenic avians and deposited into avian eggs.

In one aspect, the invention is drawn to proteins (e.g., human proteins)such as cytokines produced in avians. In a particular aspect, theinvention is drawn to human erythropoietin with a glycosylation pattern(e.g., poultry derived erythropoietin) wherein the erythropoietin isobtained from avian cells of a transgenic chicken, transgenic quail ortransgenic turkey. Also included in the invention are human proteinsincluding cytokines such as erythropoietin produced in avians inisolated or purified form and present in pharmaceutical compositions.The isolation of the recombinant proteins of the invention includingerythropoietin can be accomplished by methodologies readily apparent toa practitioner skilled in the art of protein purification. The make-upof formulations useful for producing pharmaceutical compositions arealso well known in the art. In one embodiment, the proteins of theinvention including erythropoietin have a glycosylation pattern that isobtained from poultry or avian oviduct cells, for example, tubular glandcells (e.g., tubular gland cells of a chicken).

One aspect of the present invention provides methods for producingexogenous proteins in specific tissues of avians. Exogenous proteins maybe expressed in the oviduct, blood and/or other cells and tissues of theavian. In one embodiment, transgenes are introduced into embryonicblastodermal cells, for example, near stage X, to produce a transgenicavian, such that the protein of interest is expressed in the tubulargland cells of the magnum of the oviduct, secreted into the lumen, anddeposited into the egg white of a hard shell egg. A transgenic avian soproduced can carry the transgene in its germ line. The exogenous genescan therefore be transmitted to avians by both artificial introductionof the exogenous gene into avian embryonic cells, and by thetransmission of the exogenous gene to the avian's offspring stably in aMendelian fashion.

The present invention encompasses methods of producing exogenous proteinin an avian oviduct. The methods may include a first step of providing avector that contains a coding sequence and a promoter operably linked tothe coding sequence, so that the promoter can effect expression of thenucleic acid in the avian oviduct. Next, transgenic cells and/or tissuescan be produced, wherein the vector is introduced into avian embryonicblastodermal cells, either freshly isolated, in culture, or in anembryo, so that the vector sequence is inserted, for example, randomlyinserted into the avian genome. Finally, a mature transgenic avian whichexpresses the exogenous protein in its oviduct can be derived from thetransgenic cells and/or tissue. This method can also be used to producean avian egg which contains exogenous protein such as a pharmaceuticalprotein (e.g., a cytokine) when the exogenous protein that is expressedin the oviduct is also secreted into the oviduct lumen and depositedinto the egg, for example, in the egg white of a hard shell egg.

In one aspect, the production of a transgenic bird by chromosomalinsertion of a vector into its avian genome may optionally involve DNAtransfection of embryonic blastodermal cells which are then injectedinto the subgerminal cavity beneath a recipient blastoderm. The vectorused in such a method may have a promoter which is fused to an exogenouscoding sequence and directs expression of the coding sequence in thetubular gland cells of the oviduct.

In another aspect of the invention, a random chromosomal insertion andthe production of a transgenic avian is accomplished by transduction ofembryonic blastodermal cells with replication-defective orreplication-competent retroviral particles carrying the transgenegenetic code between the 5′ and 3′ LTRs of the retroviral rector. Forinstance, an avian leukosis virus (ALV) retroviral vector or a murineleukemia virus (MLV) retroviral vector may be used which comprises amodified pNLB plasmid containing an exogenous gene that is inserteddownstream of a segment of a promoter region. An RNA copy of themodified retroviral vector, packaged into viral particles, can be usedto infect embryonic blastoderms which develop into transgenic avians.Alternatively, helper cells which produce the retroviral transducingparticles are delivered to the embryonic blastoderm.

Another aspect of the invention provides a vector which includes acoding sequence and a promoter in operational and positionalrelationship such that the coding sequence is expressed in an avianoviduct. Such vectors include, but are not limited to, an avian leukosisvirus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviralvector, and a lentivirus vector. In addition, the vector may be anucleic acid sequence which includes an LTR of an avian leukosis virus(ALV) retroviral vector, a murine leukemia virus (MLV) retroviralvector, or a lentivirus vector. The promoter is sufficient for effectingexpression of the coding sequence in the avian oviduct. The codingsequence codes for an exogenous protein which is deposited into the eggwhite of a hard shell egg. As such, the coding sequence codes forexogenous proteins such as transgenic poultry derived proteins such asinterferon-α 2b (TPD IFN-α 2b) and transgenic poultry derivederythropoietin (TPD EPO) and transgenic poultry derived granulocytecolony stimulating factor (TPD G-CSF). In one embodiment, vectors usedin the methods of the invention contain a promoter which is particularlysuited for expression of exogenous proteins in avians and their eggs. Assuch, expression of the exogenous coding sequence may occur in theoviduct and blood of the transgenic avian and in the egg white of itsavian egg. The promoters include, but are not limited to, acytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma virus(RSV) promoter, a β-actin promoter (e.g., a chicken β-actin promoter) amurine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV)promoter, an ovalbumin promoter, a lysozyme promoter, a conalbuminpromoter, an ovomucoid promoter, an ovomucin promoter, and anovotransferrin promoter. Optionally, the promoter may be a segment of atleast one promoter region, such as a segment of the ovalbumin-,lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and ovotransferrinpromoter region. In one embodiment, the promoter is a combination or afusion of one or more promoters or a fusion of a portion of one or morepromoters such as ovalbumin-, lysozyme-, conalbumin-, ovomucoid-,ovomucin-, and ovotransferrin promoters.

One aspect of the invention involves truncating the ovalbumin promoterand/or condensing the critical regulatory elements of the ovalbuminpromoter so that it retains sequences required for expression in thetubular gland cells of the magnum of the oviduct, while being smallenough that it can be readily incorporated into vectors. For instance, asegment of the ovalbumin promoter region may be used. This segmentcomprises the 5′-flanking region of the ovalbumin gene. The total lengthof the ovalbumin promoter segment may be from about 0.88 kb to about 7.4kb in length, and is preferably from about 0.88 kb to about 1.4 kb inlength. The segment preferably includes both the steroid-dependentregulatory element and the negative regulatory element of the ovalbumingene. The segment optionally also includes residues from the5′untranslated region (5′UTR) of the ovalbumin gene. Alternatively, thepromoter may be a segment of the promoter region of the lysozyme-,conalbumin-, ovomucin-, ovomucoid- and ovotransferrin genes. An exampleof such a promoter is the synthetic MDOT promoter which is comprised ofelements from the ovomucoid (MD) and ovotransferrin (OT) promoter.

In another aspect of the invention, the vectors integrated into theavian genome contain constitutive promoters which are operably linked tothe exogenous coding sequence (e.g., cytomegalovirus (CMV) promoter,rous-sarcoma virus (RSV) promoter, and a murine leukemia virus (MLV)promoter. Alternatively, a non-constitutive promoter such as a mousemammary tumor virus (MMTV) promoter may be used.

Other aspects of the invention provide for transgenic avians which carrya transgene in the genetic material of their germ-line tissue. Morespecifically, the transgene includes an exogenous gene and a promoter inoperational and positional relationship to express the exogenous gene.The exogenous gene may be expressed in the avian oviduct and in theblood of the transgenic avian. The exogenous gene codes for exogenousproteins such as pharmaceutical proteins including cytokines such as TPDIFN-α (e.g., IFN-α 2) and TPD EPO and TPD G-CSF. The exogenous proteinis deposited into the egg white of a hard shell egg.

Another aspect of the invention provides for an avian egg which containsprotein exogenous to the avian species. Use of the invention allows forexpression of exogenous proteins in oviduct cells with secretion of theproteins into the lumen of the oviduct magnum and deposition into theegg white of the avian egg. Proteins packaged into eggs may be presentin quantities of up to one gram or more per egg. The exogenous proteinincludes, but is not limited to, TPD IFN-α 2 and TPD EPO and TPD G-CSF.

Still another aspect of the invention provides an isolatedpolynucleotide sequence comprising the optimized coding sequence ofhuman interferon-α 2b (IFN-α 2b), i.e., recombinant transgenic poultryderived interferon-α 2b coding sequence which codes for transgenicpoultry derived interferon-α 2b (TPD IFN-α 2b). The invention alsoencompasses an isolated protein comprising the polypeptide sequence ofTPD IFN-α 2b, wherein the protein is O-glycosylated at Thr-106 withN-Acetyl-Galactosamine, Galactose, N-Acetyl-Glucosamine, Sialic acid,and combinations thereof.

The invention further contemplates a pharmaceutical compositioncomprising the polypeptide sequence of TPD IFN-α 2b, wherein the proteinis O-glycosylated at Thr-106 with N-Acetyl-Galactosamine, Galactose,N-Acetyl-Glucosamine, Sialic acid, and combinations thereof.

One aspect of the invention provides for coding sequences for exogenousproteins produced as disclosed herein wherein the coding sequence iscodon optimized for expression in an avian, for example, in a chicken.Codon optimization may be determined from the codon usage of at leastone, and preferably more than one, protein expressed in an avian cell(e.g., a chicken cell). For example, the codon usage may be determinedfrom the nucleic acid sequences encoding the proteins ovalbumin,lysozyme, ovomucin and ovotransferrin of chicken. For example, the DNAcoding sequence for the exogenous protein may be codon optimized usingthe BACKTRANSLATE® program of the Wisconsin Package, version 9.1(Genetics Computer Group, Inc., Madison, Wis.) with a codon usage tablecompiled from the chicken (Gallus gallus) ovalbumin, lysozyme,ovomucoid, and ovotransferrin proteins.

One aspect of the invention provides an isolated polynucleotide sequencecomprising the optimized coding sequence of human erythropoietin (EPO),i.e., recombinant transgenic poultry derived erythropoietin codingsequence which codes for transgenic poultry derived erythropoietin (TPDEPO).

Another aspect of the invention provides for a vector comprising a firstand second coding sequence and a promoter in operational and positionalrelationship to the first and second coding sequence to express thefirst and second coding sequence in an avian oviduct. In this aspect,the vector may include an internal ribosome entry site (IRES) elementpositioned between the first and second coding sequence, wherein thefirst coding sequence codes for protein X and the second coding sequencecodes for protein Y, and wherein one or both of protein X and protein Yare deposited into the egg (e.g., egg white) of a hard shell egg.

For example, protein X may be a light chain (LC) of a monoclonalantibody and protein Y may be a heavy chain (HC) of a monoclonalantibody. Alternatively, the protein encoded by the second codingsequence (e.g., enzyme) may be capable of providing post-translationalmodification of the protein encoded by the first coding sequence. Thevector optionally includes additional coding sequences and additionalIRES elements, such that each coding sequence in the vector is separatedfrom another coding sequence by an IRES element. Other examples ofemploying an IRES which are contemplated for use in the presentinvention are disclosed in, for example, U.S. patent application Ser.No. 11/047,184, filed Jan. 31, 2005, the disclosure of which isincorporated in its entirety herein by reference.

The invention also contemplates methods of producing an avian egg whichcontains proteins such as pharmaceutical proteins including monoclonalantibodies, enzymes and other proteins. Such methods may includeproviding a vector with a promoter, coding sequences, and at least oneIRES element; creating transgenic cells or tissue by introducing thevector into avian embryonic blastodermal cells, wherein the vectorsequence is randomly inserted into the avian genome; and deriving amature transgenic avian from the transgenic cells or tissue. Thetransgenic avian so derived may express the coding sequences in itsoviduct, and the resulting protein secreted into the oviduct lumen, sothat the protein is deposited into the egg white of a hard shell egg. Inaddition, the invention includes progeny of the transgenic avians whichproduce eggs containing the recombinant protein. Typically, the progenywill either contain the transgene in essentially all the cells of thebird or none of the cells of the progeny bird will contain thetransgene.

One important aspect of the present invention relates to avian hardshell eggs (e.g., chicken hard shell eggs) which contain an exogenouspeptide or protein including, but not limited to, a pharmaceuticalprotein. The exogenous peptide or protein may be encoded by a transgeneof a transgenic avian. In one embodiment, the exogenous peptide orprotein (e.g., pharmaceutical protein) is glycosylated. The protein maybe present in any useful amount. In one embodiment, the protein ispresent in an amount in a range of between about 0.01 μg per hard-shellegg and about 1 gram per hard-shell egg. In another embodiment, theprotein is present in an amount in a range of between about 1 μg perhard-shell egg and about 1 gram per hard-shell egg. For example, theprotein may be present in an amount in a range of between about 10 μgper hard-shell egg and about 1 gram per hard-shell egg (e.g., a range ofbetween about 10 μg per hard-shell egg and about 400 milligrams perhard-shell egg).

In one embodiment, the exogenous protein of the invention, for example,the exogenous pharmaceutical protein, is present in the egg white of theegg. In one embodiment, the protein is present in an amount in a rangeof between about 1 ng per milliliter of egg white and about 0.2 gram permilliliter of egg white. For example, the protein may be present in anamount in a range of between about 0.1 μg per milliliter of egg whiteand about 0.2 gram per milliliter of egg white (e.g., the protein may bepresent in an amount in a range of between about 1 μg per milliliter ofegg white and about 100 milligrams per milliliter of egg white. In oneembodiment, the protein is present in an amount in a range of betweenabout 1 μg per milliliter of egg white and about 50 milligrams permilliliter of egg white. For example, the protein may be present in anamount in a range of about 1 μg per milliliter of egg white and about 10milligrams per milliliter of egg white (e.g., the protein may be presentin an amount in a range of between about 1 μg per milliliter of eggwhite and about 1 milligrams per milliliter of egg white). In oneembodiment, the protein is present in an amount of more than 0.1 μg permilliliter of egg white. In one embodiment, the protein is present in anamount of more than 0.5 μg per milliliter of egg white. In oneembodiment, the protein is present in an amount of more than 1 μg permilliliter of egg white. In one embodiment, the protein is present in anamount of more than 1.5 μg per milliliter of egg white.

The invention contemplates the production of hard shell eggs containingany useful protein including one or more pharmaceutical proteins. Suchproteins include, but are not limited to, hormones, immunoglobulins orportions of immunoglobulins, cytokines (e.g., GM-CSF, G-CSF,erythropoietin and interferon) and CTLA4. The invention also includesthe production of hard shell eggs containing fusion proteins including,but not limited to, immunoglobulins or portions of immunoglobulins fusedto certain useful peptide sequences. In one embodiment, the inventionprovides for the production of hard shell eggs containing an antibody Fcfragment. For example, the eggs may contain an Fc-CTLA4 fusion proteinin accordance with the invention.

The avians developed from the blastodermal cells into which the vectorhas been introduced are the G0 generation and can be referred to as“founders”. Founder birds are typically chimeric for each insertedtransgene. That is, only some of the cells of the G0 transgenic birdcontain the transgene(s). The G0 generation typically is also hemizygousfor the transgene(s). The G0 generation may be bred to non-transgenicanimals to give rise to G1 transgenic offspring which are alsohemizygous for the transgene and contain the transgene(s) in essentiallyall of the bird's cells. The G1 hemizygous offspring may be bred tonon-transgenic animals giving rise to G2 hemizygous offspring or may bebred together to give rise to G2 offspring homozygous for the transgene.Substantially all of the cells of birds which are positive for thetransgene that are derived from G1 offspring will contain thetransgene(s). In one embodiment, hemizygotic G2 offspring from the sameline can be bred to produce G3 offspring homozygous for the transgene.In one embodiment, hemizygous G0 animals are bred together to give riseto homozygous G1 offspring containing two copies of the transgene(s) ineach cell of the animal. These are merely examples of certain usefulbreeding methods and the present invention contemplates the employmentof any useful breeding method such as those known to individuals ofordinary skill in the art.

One aspect of the invention is directed to compositions which containproteins produced in accordance with the invention that have a poultryderived glycosylation pattern, such as a chicken derived glycosylationpattern. One aspect of the invention is directed to compositions whichcontain proteins produced in accordance with the invention that have anavian derived glycosylation pattern, such as a chicken derivedglycosylation pattern. For example, the invention includespharmaceutical proteins having a poultry derived glycosylation patternsuch as one or more of the glycosylation patterns disclosed herein. Theinvention also includes human proteins having a poultry derivedglycosylation pattern such as one or more of the glycosylation patternsdisclosed herein.

In one aspect, the invention includes G-CSF wherein the G-CSF has apoultry derived glycosylation pattern, i.e., a transgenic poultryderived G-CSF or TPD G-CSF. In one aspect, the invention includes G-CSFwherein the G-CSF has a transgenic avian derived glycosylation pattern,i.e., a transgenic avian derived G-CSF. In one embodiment, theglycosylation pattern is other than that of G-CSF produced in a humancell and/or in a CHO cell. That is, the compositions have a G-CSFmolecule with a poultry or avian derived carbohydrate chain (i.e.,glycosylation structure) and that carbohydrate chain or glycosylationstructure is not found on G-CSF obtained from human cells and/or CHOcells. However, the composition may also include G-CSF molecules thathave glycosylation structures that are the same as that found on G-CSFobtained from CHO cells and/or human cells. Glycosylation of human G-CSFproduced in CHO cells is disclosed in Holloway, C. J., European J. ofCancer (1994) vol 30A, pS2-S6, the disclosure of which is incorporatedin its entirety herein by reference; in Oheda et al (1988) J. Biochem.,v 103, p 544-546, the disclosure of which is incorporated in itsentirety herein by reference and in Andersen et al (1994) Glycobiology,vol 4, p 459-467, the disclosure of which is incorporated in itsentirety herein by reference. It appears that structures such as A and Gshown in Example 20 may be the same or similar to glycosylationstructures reported for G-CSF produced in CHO cells. In one embodiment,the glycosylation pattern of the G-CSF produced in accordance with theinvention is other than that of G-CSF produced in mammalian cell.

In one embodiment, the invention provides for the G-CSF to be isolated.That is, the G-CSF contained in the composition may be an isolatedG-CSF. For example, the G-CSF may be isolated from egg white. Theisolated G-CSF may be G-CSF molecules having differing glycosylationstructures among the G-CSF molecules or the isolated G-CSF may be anisolated individual species of G-CSF molecules having only oneparticular glycosylation structure among the species of G-CSF molecules.

In one embodiment, the G-CSF of a composition of the invention ispresent in a hard shell egg. For example, the G-CSF may be present inthe egg white of a hard shell egg laid by a transgenic avian of theinvention. That is, in one embodiment, the invention is directed toavian (e.g., chicken) egg white containing G-CSF of the invention. Inone embodiment, the G-CSF is present in the egg white in an amount inexcess of about 1 microgram per ml of egg white. For example, the G-CSFcan be present in an amount greater that about 2 micrograms per ml ofegg white (e.g., present in an amount of about 2 micrograms to about 200micrograms per ml of egg white).

In one particular aspect of the invention, the G-CSF is glycosylated inan oviduct cell of the avian, e.g., glycosylated in an oviduct cell of achicken. For example, the G-CSF can be produced and glycosylated in anoviduct cell. In one embodiment, the G-CSF is glycosylated in a tubulargland cell (e.g., the G-CSF is produced and glycosylated in a tubulargland cell).

The G-CSF is believed to be glycosylated at threonine 133. However, theinvention is not limited to glycosylation at any particular site on aG-CSF molecule.

Typically, the G-CSF of the invention is human G-CSF. In one embodiment,the mature G-CSF has the amino acid sequence of FIG. 18 C.

In one embodiment, compositions of the invention include G-CSF moleculesglycosylated with:

The invention is also specifically directed to compositions containingG-CSF molecules that have one of these particular glycosyationstructures. Such compositions may also include one or more G-CSFmolecules having one or more other glycosylation structures.

That is, in one embodiment, the invention is specifically directed tocompositions containing G-CSF molecules that have:

and to compositions containing G-CSF molecules that have:

and to compositions containing G-CSF molecules that have:

and to compositions containing G-CSF molecules that have:SA-Gal-NAcGal-;and to compositions containing G-CSF molecules that have:

and to compositions containing G-CSF molecules that have:

and to compositions containing G-CSF molecules that have:Gal-NAcGal-,wherein

-   Gal=Galactose,-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.

The invention is also directed to methods of increasing white blood cellcount in a patient which include administering to a patient atherapeutically effective amount of G-CSF produced in accordance withthe invention. Typically, the therapeutically effective amount is anamount of G-CSF that increases the white blood cell count in a patientby a desired amount.

One aspect of the invention relates to compositions containing EPO,i.e., EPO molecules produced in accordance with the invention. In aparticularly useful embodiment, the EPO is purified or isolated. Forexample, the EPO has been removed from the contents of a hard shell egglaid by a transgenic avian. In one particularly useful embodiment, theEPO is human EPO. In one embodiment, the EPO of the invention has aglycosylation pattern resulting from the EPO being produced in anoviduct cell of an avian. Another aspect of the invention relates tocompositions containing EPO that has a glycosylation pattern wherein theglycosylation pattern is other than that of EPO produced in a human cellor a CHO cell and the EPO is produced in an oviduct cell of a chicken.In one aspect the invention provides for compositions that containisolated EPO (e.g., human EPO) having an avian or poultry derivedglycosylation pattern. For example, the compositions can contain amixture of EPO molecules produced in avians, for example, chickens, inaccordance with the invention and isolated from egg white. In one usefulembodiment, the EPO containing compositions are pharmaceuticalformulations.

In one embodiment, the oligosaccharides present on the EPO of theinvention do not contain fucose. In another embodiment, about 90% ormore of the N-linked oligosaccharides present on the EPO of theinvention do not contain fucose. In another embodiment, about 80% ormore of the N-linked oligosaccharides present on the EPO of theinvention do not contain fucose. In another embodiment, about 70% ormore of the N-linked oligosaccharides present on the EPO of theinvention do not contain fucose. In another embodiment, about 60% ormore of the N-linked oligosaccharides present on the EPO of theinvention do not contain fucose. In another embodiment, about 50% ormore of the N-linked oligosaccharides present on the EPO of theinvention do not contain fucose.

In one embodiment, about 95% or more of the N-linked oligosaccharidespresent on the EPO of the invention do not contain sialic acid. Inanother embodiment, about 90% or more of the N-linked oligosaccharidespresent on the EPO of the invention do not contain sialic acid. Inanother embodiment, about 80% or more of the N-linked oligosaccharidespresent on the EPO of the invention do not contain sialic acid. Inanother embodiment, more than about 70% or more of the N-linkedoligosaccharides present on the EPO of the invention do not containsialic acid. In another embodiment, about 60% or more of the N-linkedoligosaccharides present on the EPO of the invention do not containsialic acid. In another embodiment, about 50% or more of the N-linkedoligosaccharides present on the EPO of the invention do not containsialic acid.

In one embodiment, about 95% or more of the N-linked oligosaccharidespresent on the EPO of the invention contain a terminal N-AcetylGlucosamine. In another embodiment, about 90% or more of the N-linkedoligosaccharides present on the EPO of the invention contain a terminalN-Acetyl Glucosamine. In another embodiment, about 80% or more of theN-linked oligosaccharides present on the EPO of the invention contain aterminal N-Acetyl Glucosamine. In another embodiment, about 70% or moreof the N-linked oligosaccharides present on the EPO of the inventioncontain a terminal N-Acetyl Glucosamine. In another embodiment, about60% or more of the N-linked oligosaccharides present on the EPO of theinvention contain a terminal N-Acetyl Glucosamine. In anotherembodiment, about 50% or more of the N-linked oligosaccharides presenton the EPO of the invention contain a terminal N-Acetyl Glucosamine.

In one embodiment, essentially none of the N-linked oligosaccharidesstructure types present on the EPO molecules of the invention containfucose. In another embodiment, about 90% or more of the N-linkedoligosaccharides structure types present on the EPO molecules of theinvention do not contain fucose. For example, if there are 20oligosaccharide structure types, then 18 or more of the structure typeswill not contain fucose. In another embodiment, about 80% or more of theN-linked oligosaccharides structure types present on the EPO moleculesof the invention do not contain fucose. In another embodiment, about 70%or more of the N-linked oligosaccharides structure types present on theEPO molecules of the invention do not contain fucose. In anotherembodiment, about 60% or more of the N-linked oligosaccharides structuretypes present on the EPO molecules of the invention do not containfucose. In another embodiment, about 50% or more of the N-linkedoligosaccharides structure types present on the EPO molecules of theinvention do not contain fucose.

In one embodiment, essentially none of the N-linked oligosaccharidesstructure types present on the EPO molecules of the invention containsialic acid. In another embodiment, about 90% or more of the N-linkedoligosaccharides structure types present on the EPO molecules of theinvention do not contain sialic acid. For example, if there are 20ologosaccharide structure types, then 18 or more of the structure typeswill not contain sialic acid. In another embodiment, about 80% or moreof the N-linked oligosaccharides structure types present on the EPOmolecules of the invention do not contain sialic acid. In anotherembodiment, about 70% or more of the N-linked oligosaccharides structuretypes present on the EPO molecules of the invention do not containsialic acid. In another embodiment, about 60% or more of the N-linkedoligosaccharides structure types present on the EPO molecules of theinvention do not contain sialic acid. In another embodiment, about 50%or more of the N-linked oligosaccharides structure types present on theEPO molecules of the invention do not contain sialic acid.

In one embodiment, all of the N-linked oligosaccharides structure typespresent on the EPO molecules of the invention contain a terminalN-Acetyl Glucosamine. In another embodiment, about 90% or more of theN-linked oligosaccharides structure types present on the EPO moleculesof the invention contain a terminal N-Acetyl Glucosamine. For example,if there are 20 oligosaccharide structure types, then 18 or more of thestructure types will contain a terminal N-Acetyl Glucosamine. In anotherembodiment, about 80% or more of the N-linked oligosaccharides structuretypes present on the EPO molecules of the invention contain a terminalN-Acetyl Glucosamine. In another embodiment, about 70% or more of theN-linked oligosaccharides structure types present on the EPO moleculesof the invention contain a terminal N-Acetyl Glucosamine. In anotherembodiment, about 60% or more of the N-linked oligosaccharides structuretypes present on the EPO molecules of the invention contain a terminalN-Acetyl Glucosamine. In another embodiment, about 50% or more of theN-linked oligosaccharides structure types present on the EPO moleculesof the invention contain a terminal N-Acetyl Glucosamine.

In one aspect, the invention is directed to EPO obtained from atransgenic avian, for example, a transgenic chicken, which contains atransgene encoding the EPO. In one embodiment, the EPO is produced in anavian oviduct cell, for example, a tubular gland cell. In oneembodiment, the EPO is contained in a hard shell egg, for example, ahard shell egg laid by an avian, e.g., a chicken. For example, the EPOmay be present in the contents of an intact hard shell egg. In oneparticularly useful embodiment, the EPO of the invention is human EPO.

In one aspect, the invention is drawn to compositions containingisolated EPO molecules, for example, human EPO molecules, wherein theEPO is produced in an avian which contains a transgene encoding the EPO.In one embodiment, the EPO is produced in an oviduct cell (e.g., atubular gland cell) of a transgenic avian (e.g., transgenic chicken) andthe EPO is isolated from egg white of the transgenic avian. In oneembodiment, the EPO of the invention has the amino acid sequence of FIG.19A. It is contemplated that the EPO is N-glycosylated and/orO-glycosylated. In one embodiment, the EPO is glycosylated in theoviduct cell (e.g., tubular gland cell) of the bird, for example, achicken.

In one aspect, the invention relates to a composition, for example, apharmaceutical formulation, containing isolated EPO, for example, humanEPO, having an avian derived glycosylation pattern. In one aspect, theinvention relates to a composition, for example, a pharmaceuticalformulation, containing isolated EPO, for example, human EPO, having apoultry derived glycosylation pattern. In one aspect, the inventionrelates to a composition, for example, a pharmaceutical formulation,containing isolated EPO, for example, human EPO, produced in accordancewith the invention. In one embodiment, EPO in compositions of theinvention contains a glycosylation pattern other than that of EPOproduced in a mammalian cell. In one embodiment, EPO in compositions ofthe invention contains a glycosylation pattern other than that of EPOproduced in a CHO cell and a human cell. In one embodiment, EPO of theinvention is attached to one or more N-linked oligosaccharide structuresdisclosed herein (e.g., those shown in FIG. 21). In one embodiment, EPOof the invention is attached to one or more O-linked oligosaccharidestructures disclosed herein (e.g., those shown in FIG. 20).

One aspect of the invention is drawn to methods of treating a patientcomprising administering to a patient a therapeutically effective amountof EPO obtained from a transgenic avian. In one embodiment, thetherapeutically effective amount is an amount that increases the redblood cell count in a patient by a desired amount. It is contemplatedthat EPO produced in accordance with the invention can be used to treatchronic kidney disease, for example, where tissues fail to sustainproduction of erythropoietin.

One aspect of the invention is drawn to compositions containing isolatedglycosylated human protein molecules produced in the oviduct of atransgenic avian wherein the transgenic avian (e.g., transgenic chicken)contains a transgene encoding the human protein and wherein the humanprotein contains a chicken derived oligosaccharide which is not normallypresent on the human protein. In one embodiment, the human protein isattached to one or more N-linked oligosaccharide structures disclosedherein (e.g., those shown in FIG. 21). In one embodiment, the humanprotein is attached to one or more O-linked oligosaccharide structuresdisclosed herein (e.g., those shown in FIG. 20).

In one embodiment, the invention is directed to isolated proteinmolecules produced in the oviduct of a transgenic chicken, for example,as disclosed herein, wherein the transgenic chicken contains a transgeneencoding the protein molecule and wherein the protein molecule containsa chicken derived oligosaccharide. For example, the protein molecule canbe a glycosylated form of GM-CSF, interferon β, fusion protein, CTLA4-Fcfusion protein, growth hormones, cytokines, structural, interferon,lysozyme, β-casein, albumin, α-1 antitrypsin, antithrombin III,collagen, factors VIII, IX, X (and the like), fibrinogen, lactoferrin,protein C, tissue-type plasminogen activator (tPA), somatotropin, andchymotrypsin, immunoglobulins, antibodies, immunotoxins, factor VIII,b-domain deleted factor VIII, factor VIIa, factor IX, anticoagulants;hirudin, alteplase, tpa, reteplase, tpa, tpa-3 of 5 domains deleted,insulin, insulin lispro, insulin aspart, insulin glargine, long-actinginsulin analogs, glucagons, tsh, follitropin-beta, fsh, pdgh, inf-beta1b, ifn-beta 1a, ifn-gamma1b, il-2, il-11, hbsag, ospa, dornase-alphadnase, beta glucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusionprotein, tnfr-lgg fragment fusion protein laronidase, dnaases,alefacept, tositumomab, murine mab, alemtuzumab, rasburicase, agalsidasebeta, teriparatide, parathyroid hormone derivatives, adalimumab (lgg1),anakinra, biological modifier, nesiritide, human b-type natriureticpeptide (hbnp), colony stimulating factors, pegvisomant, human growthhormone receptor antagonist, recombinant activated protein c,omalizumab, immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH,glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone,pigmentary hormones, somatomedin, luteinizing hormone, chorionicgonadotropin, hypothalmic releasing factors, etanercept, antidiuretichormones, prolactin and thyroid stimulating hormone, an immunoglobulinpolypeptide, immunoglobulin polypeptide D region, immunoglobulinpolypeptide J region, immunoglobulin polypeptide C region,immunoglobulin light chain, immunoglobulin heavy chain, animmunoglobulin heavy chain variable region, an immunoglobulin lightchain variable region and a linker peptide. Proteins not normallyglycosylated can be engineered to contain a glycosylation site whichwill be glycosylated in the avian system, as is understood by apractitioner of skill in the art. In one embodiment, the isolatedprotein has attached one or more N-linked oligosaccharide structuresdisclosed herein (e.g., those shown in FIG. 21). In one embodiment, theisolated protein is attached to one or more O-linked oligosaccharidestructures disclosed herein (e.g., those shown in FIG. 20).

Features (e.g., compositions, glycosylation structures) specificallycontemplated for certain proteins disclosed herein such as EPO are alsocontemplated for other specific proteins disclosed herein, which can beproduced in accordance with the invention.

The invention also includes, methods of making glycosylated proteinsdisclosed herein such as erythropoietin comprising producing atransgenic avian which contains a transgene encoding protein (e.g.,erythropoietin) wherein the protein is packaged into a hard shell egglaid by the avian. Also included are the eggs laid by the avians whichcontain the protein (e.g., erythropoietin).

The invention also provides for compositions which contain isolatedmixtures of an individual type of useful protein molecule, such as thoseproteins disclosed herein, where one or more of the protein moleculescontained in the mixture has a specific oligosaccharide structureattached, in particular an oligosaccharide structure disclosed hereinwhich may be produced by a transgenic avian. For example, the inventionprovides for isolated mixtures of EPO molecules, for example, human EPOmolecules (e.g., EPO of SEQ ID NO: 50) which contain an EPO moleculeglycosylated with one or more of:

and each of the other oligosaccharide structure shown in FIG. 20 andFIG. 21.

Any useful combination of features described herein is included withinthe scope of the present invention provided that the features includedin any such combination are not mutually inconsistent as will beapparent from the context, this specification, and the knowledge of oneof ordinary skill in the art.

Additional objects and aspects of the present invention will become moreapparent upon review of the detailed description set forth below whentaken in conjunction with the accompanying figures, which are brieflydescribed as follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate ovalbumin promoter expression vectorscomprising ovalbumin promoter segments and a coding sequence, gene X,which encodes an exogenous protein X. X represents any exogenous gene orexogenous protein of interest.

FIGS. 2A, 2B, 2C and 2D illustrate retroviral vectors of the inventioncomprising an ovalbumin promoter and a coding sequence, gene X, encodingan exogenous protein X. X represents any exogenous gene or exogenousprotein of interest.

FIG. 2E illustrates a method of amplifying an exogenous gene forinsertion into the vectors of 2A and 2B.

FIG. 2F illustrates a retroviral vector comprising an ovalbumin promotercontrolling expression of a coding sequence, gene X, and an internalribosome entry site (IRES) element enabling expression of a secondcoding sequence, gene Y. X and Y represent any gene of interest.

FIGS. 3A and 3B show schematic representations of the ALV-derivedvectors pNLB and pNLB-CMV-BL, respectively. Because NLB has not beensequenced in its entirety, measurements in bp (base pair) are estimatedfrom published data (Cosset et al., 1991; Thoraval et al., 1995) anddata discussed herein. The vectors are both shown as they would appearwhile integrated into the chicken genome.

FIGS. 4A and 4B show the amount of β-lactamase (lactamase) in the bloodserum of chimeric and transgenic chickens. In FIG. 4A the concentrationof bioactive lactamase in the serum of G0 chickens transduced with theNLB-CMV-BL transgene was measured at 8 month post-hatch. The generation,sex and wing band numbers are indicated. Lactamase serum concentrationswere measured for G1 transgenic chickens at 6 to 7 months post-hatch.Arrows indicate G1 chickens bred from rooster 2395. In FIG. 4B thelactamase serum concentration was measured for G1 and G2 transgenicchickens. Arrows indicate G2s bred from hen 5657 or rooster 4133.Samples from chickens 4133, 5308, and 5657 are the same as those in FIG.4A. Samples from G2 birds bred from 5657 were collected at 3 to 60 dayspost-hatch. Samples from G2 birds bred from 4133 were collected at 3month post-hatch.

FIG. 5 shows the pedigree of chickens containing the transgenic lociharbored by hen 5657 (FIG. 5A) or rooster 4133 (FIG. 5B). 2395 was arooster that carried multiple transgenic loci. 2395 was bred to anon-transgenic hen, yielding 3 offspring each carrying the transgene ina unique position of the chicken genome. For simplicity, transgenicprogeny for which expression data were not shown as well asnon-transgenic progeny were omitted from the pedigree. Band numbers areindicated by the following symbols: ∘ hen; □ rooster; ● hen carrying theNLB-CMV-BL transgene; ▪ rooster carrying the NLB-CMV-BL transgene.

FIG. 6 shows β-lactamase (lactamase) in the egg white of hen 5657 andher offspring. In FIG. 6A egg white from hen 5657 and her transgenicoffspring were assayed for active lactamase. The control is fromuntreated hens and clutchmate is a non-transgenic G2 bred from hen 5657.Eggs were collected in March 2000. Arrows indicate G2s bred from hen5657. In FIG. 6B egg white samples from G2 transgenic hens carrying onecopy of the transgene (hemizygous) were compared with that of G3 hen6978 which harbored two copies (homozygous). Eggs were collected inFebruary 2001. The generation and wing band numbers are indicated to theleft.

FIG. 7 shows β-lactamase (lactamase) in the eggs of G2 and G3 hens bredfrom rooster 4133. In FIG. 7A egg whites from four representativehemizygous transgenic hens bred from rooster 4133 were assayed foractive lactamase. Eggs were collected in October 1999, March 2000 andFebruary 2001 and a minimum of 4 eggs per hen were assayed one monthafter each set was collected. The control represents egg white fromuntreated hens. Band numbers are indicated to the left. The average ofthe 4 hens for each period is calculated. In FIG. 7B egg white fromhemizygous G2 transgenic hens were compared with that of hemizygous andhomozygous transgenic G3 hens. The eggs were collected in February 2001.The generation and transgene copy number are displayed in the data barfor each hen. The average concentration for hens carrying one or twocopies is at the bottom of the chart.

FIGS. 8A and 8B show the pNLB-CMV-IFN vector for expressing IFN-α 2b inchickens; and the pNLB-MDOT-EPO vector used for expressingerythropoietin (EPO) in chickens, respectively.

FIG. 9 depicts the novel glycosylation pattern of transgenic poultryderived interferon-α 2b (TPD IFN-α 2b), including all 6 bands.

FIG. 10 shows the comparison of human peripheral blood leukocyte derivedinterferon-α 2b (PBL IFN-α 2b or natural hIFN) and transgenic poultryderived interferon-α 2b (TPD IFN-α 2b or egg white hIFN).

FIG. 11A depicts the synthetic nucleic acid sequence (cDNA, residues1-498) of optimized human interferon-α 2b (IFN-α 2b), i.e., recombinantTPD IFN-α 2b (SEQ ID NO: 1). FIG. 11B depicts the synthetic amino acidsequence (residues 1-165) of transgenic poultry derived interferon-α 2b(TPD IFN-α 2b) (SEQ ID NO: 2).

FIG. 12A depicts the synthetic nucleic acid sequence (cDNA, residues1-579) of optimized human erythropoietin (EPO) i.e., recombinant TPD EPO(SEQ ID NO: 3). FIG. 12B depicts the synthetic amino acid sequence(residues 1-193) of transgenic poultry derived erythropoietin (TPD EPO)(SEQ ID NO: 4). (For natural human EPO see also NCBI Accession Number NP000790).

FIG. 13 shows the synthetic MDOT promoter linked to the IFN-MM CDS. TheMDOT promoter contains elements from the chicken ovomucoid gene(ovomucoid promoter) ranging from −435 to −166 bp (see NCBI AccessionNumber J00894) and the chicken conalbumin gene (ovotransferrin promoter)ranging from −251 to +29 bp (see NCBI Accession Numbers Y00497, M11862and X01205).

FIG. 14 provides a summary of the major egg white proteins.

FIGS. 15A and 15D show the pCMV-LC-emcvIRES-HC vector, wherein the lightchain (LC) and heavy chain (HC) of a human monoclonal antibody wereexpressed from this single vector by placement of an IRES from theencephalomyocarditis virus (EMCV) in order to test for expression ofmonoclonal antibodies. In comparison, FIGS. 15B and 15C show theseparate vectors pCMV-HC and pCMV-LC, respectively, wherein thesevectors were also used to test for expression of monoclonal antibodies.

FIG. 16 shows a silver stained SDS PAGE of Neupogen® (lane A) and TPDG-CSF (lane B).

FIG. 17 depicts the increase in Absolute Neutrophil Count (ANC) of TPDG-CSF compared to bacterial derived human G-CSF over a 14 day period.

FIG. 18A (SEQ ID NO: 39) shows the nucleotide sequence encoding theamino acid sequence of FIG. 18B. FIG. 18 B (SEQ ID NO: 40), whichcorresponds to NCBI Accession NP 7577373, shows the amino acid sequenceof G-CSF including the natural signal sequence which is cleaved away toform the mature G-CSF during cellular secretion. FIG. 18C (SEQ ID NO:41) shows the amino acid sequence of the mature G-CSF protein producedin accordance with the present invention.

FIG. 19A shows the nucleotide coding sequence used to produce the 165amino acid form of human erythropoietin in transgenic avians. FIG. 19Bshows the amino acid sequence of the 165 amino acid form of humanerythropoietin produced in transgenic avians.

FIG. 20 shows representative O-linked glycosylation structuresdetermined for the erythropoietin produced in accordance with theinvention.

FIG. 21A and FIG. 21B shows representative N-linked glycosylationstructures determined for the erythropoietin produced in accordance withthe invention. The bracket in front of a group of sugar residues meansthat the indicated sugar(s) can be attached to any of the bracketedsugars. For example, in Structure E-n the indicated galactose moleculeattached to a sialic acid can be attached to any one of the fiveterminal n-acetyl glucosamines. Postulated linkages are also shown forthe structures, as is understood in the art. It is contemplated that foreach of the structures indicated as C-n, E-n, F-n and H-n, the twoterminal NAcGlu residues linked to a single mannose may be 2,6-linked tomannose instead of 2,4 linked to the mannose.

FIG. 22 shows the in vitro activity of the purified transgenic chickenderived EPO. ED50=0.44 ng/ml.

DETAILED DESCRIPTION

Certain definitions are set forth herein to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

A “nucleic acid or polynucleotide sequence” includes, but is not limitedto, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNAsequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more modified bases.

The term “avian” as used herein refers to any species, subspecies orrace of organism of the taxonomic class ava, such as, but not limited tochicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks,crows and ratites including ostrich, emu and cassowary. The termincludes the various known strains of Gallus gallus, or chickens, (forexample, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, NewHampshire, Rhode Island, Australorp, Minorca, Amrox, California Gray),as well as strains of turkeys, pheasants, quails, duck, ostriches andother poultry commonly bred in commercial quantities. It also includesan individual avian organism in all stages of development, includingembryonic and fetal stages.

“Therapeutic proteins” or “pharmaceutical proteins” include an aminoacid sequence which in whole or in part makes up a drug.

A “coding sequence” or “open reading frame” refers to a polynucleotideor nucleic acid sequence which can be transcribed and translated (in thecase of DNA) or translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by atranslation start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A transcription terminationsequence will usually be located 3′ to the coding sequence. A codingsequence may be flanked on the 5′ and/or 3′ ends by untranslatedregions.

“Exon” refers to that part of a gene which, when transcribed into anuclear transcript, is “expressed” in the cytoplasmic mRNA after removalof the introns or intervening sequences by nuclear splicing.

Nucleic acid “control sequences” or “regulatory sequences” refer topromoter sequences, translational start and stop codons, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, asnecessary and sufficient for the transcription and translation of agiven coding sequence in a defined host cell. Examples of controlsequences suitable for eukaryotic cells are promoters, polyadenylationsignals, and enhancers. All of these control sequences need not bepresent in a recombinant vector so long as those necessary andsufficient for the transcription and translation of the desired gene arepresent.

“Operably or operatively linked” refers to the configuration of thecoding and control sequences so as to perform the desired function.Thus, control sequences operably linked to a coding sequence are capableof effecting the expression of the coding sequence. A coding sequence isoperably linked to or under the control of transcriptional regulatoryregions in a cell when DNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA that can be translated intothe encoded protein. The control sequences need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The terms “heterologous” and “exogenous” as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct or with a particular chromosomal locus, and/or arenot normally associated with a particular cell. Thus, an “exogenous”region of a nucleic acid construct is an identifiable segment of nucleicacid within or attached to another nucleic acid molecule that is notfound in association with the other molecule in nature. For example, anexogenous region of a construct could include a coding sequence flankedby sequences not found in association with the coding sequence innature. Another example of an exogenous coding sequence is a constructwhere the coding sequence itself is not found in nature (e.g., syntheticsequences having codons different from the native gene). Similarly, ahost cell transformed with a construct or nucleic acid which is notnormally present in the host cell would be considered exogenous forpurposes of this invention.

As used herein the terms “oligosaccharide”, “oligosaccharide structure”,“glycosylation pattern” and “glycosylation structure” have essentiallythe same meaning and each refer to one or more structures which areformed from sugar residues and are attached to glycosylated proteins.

“Exogenous protein” as used herein refers to a protein not naturallypresent in a particular tissue or cell, a protein that is the expressionproduct of an exogenous expression construct or transgene, or a proteinnot naturally present in a given quantity in a particular tissue orcell. A protein that is exogenous to an egg is a protein that is notnormally found in the egg. For example, a protein exogenous to an eggmay be a protein that is present in the egg as a result of theexpression of a coding sequence present in a transgene of the animallaying the egg.

“Endogenous gene” refers to a naturally occurring gene or fragmentthereof normally associated with a particular cell.

“EPO” means “erythropoietin” and the two terms are used interchangeablythroughout the specification.

The expression products described herein may consist of proteinaceousmaterial having a defined chemical structure. However, the precisestructure depends on a number of factors, particularly chemicalmodifications common to proteins. For example, since all proteinscontain ionizable amino and carboxyl groups, the protein may be obtainedin acidic or basic salt form, or in neutral form. The primary amino acidsequence may be derivatized using sugar molecules (glycosylation) or byother chemical derivatizations involving covalent or ionic attachmentwith, for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro or in vivo, the latter being performed by a host cellthrough post-translational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Sambrook, Fritsch, and Maniatis, MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989).

“Vector” means a polynucleotide comprised of single strand, doublestrand, circular, or supercoiled DNA or RNA. A typical vector may becomprised of the following elements operatively linked at appropriatedistances for allowing functional gene expression: replication origin,promoter, enhancer, 5′ mRNA leader sequence, ribosomal binding site,nucleic acid cassette, termination and polyadenylation sites, andselectable marker sequences. One or more of these elements may beomitted in specific applications. The nucleic acid cassette can includea restriction site for insertion of the nucleic acid sequence to beexpressed. In a functional vector the nucleic acid cassette contains thenucleic acid sequence to be expressed including translation initiationand termination sites. An intron optionally may be included in theconstruct, for example, 5′ to the coding sequence. A vector isconstructed so that the particular coding sequence is located in thevector with the appropriate regulatory sequences, the positioning andorientation of the coding sequence with respect to the control sequencesbeing such that the coding sequence is transcribed under the “control”of the control or regulatory sequences. Modification of the sequencesencoding the particular protein of interest may be desirable to achievethis end. For example, in some cases it may be necessary to modify thesequence so that it may be attached to the control sequences with theappropriate orientation; or to maintain the reading frame. The controlsequences and other regulatory sequences may be ligated to the codingsequence prior to insertion into a vector. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site whichis in reading frame with and under regulatory control of the controlsequences.

A “promoter” is a site on the DNA to which RNA polymerase binds toinitiate transcription of a gene. In some embodiments the promoter willbe modified by the addition or deletion of sequences, or replaced withalternative sequences, including natural and synthetic sequences as wellas sequences which may be a combination of synthetic and naturalsequences. Many eukaryotic promoters contain two types of recognitionsequences: the TATA box and the upstream promoter elements. The former,located upstream of the transcription initiation site, is involved indirecting RNA polymerase to initiate transcription at the correct site,while the latter appears to determine the rate of transcription and isupstream of the TATA box. Enhancer elements can also stimulatetranscription from linked promoters, but many function exclusively in aparticular cell type. Many enhancer/promoter elements derived fromviruses, e.g., the SV40 promoter, the cytomegalovirus (CMV) promoter,the rous-sarcoma virus (RSV) promoter, and the murine leukemia virus(MLV) promoter are all active in a wide array of cell types, and aretermed “ubiquitous”. Alternatively, non-constitutive promoters such asthe mouse mammary tumor virus (MMTV) promoter may also be used in thepresent invention. The nucleic acid sequence inserted in the cloningsite may have any open reading frame encoding a polypeptide of interest,with the proviso that where the coding sequence encodes a polypeptide ofinterest, it should lack cryptic splice sites which can block productionof appropriate mRNA molecules and/or produce aberrantly spliced orabnormal mRNA molecules.

The term “poultry derived” refers to a composition or substance producedby or obtained from poultry. “Poultry” refers to birds that can be keptas livestock, including but not limited to, chickens, duck, turkey,quail and ratites. For example, “poultry derived” may refer to chickenderived, turkey derived and/or quail derived.

A “marker gene” is a gene which encodes a protein that allows foridentification and isolation of correctly transfected cells. Suitablemarker sequences include, but are not limited to green, yellow, and bluefluorescent protein genes (GFP, YFP, and BFP, respectively). Othersuitable markers include thymidine kinase (tk), dihydrofolate reductase(DHFR), and aminoglycoside phosphotransferase (APH) genes. The latterimparts resistance to the aminoglycoside antibiotics, such as kanamycin,neomycin, and geneticin. These, and other marker genes such as thoseencoding chloramphenicol acetyltransferase (CAT), β-lactamase,β-galactosidase (β-gal), may be incorporated into the primary nucleicacid cassette along with the gene expressing the desired protein, or theselection markers may be contained on separate vectors andcotransfected.

A “reporter gene” is a marker gene that “reports” its activity in a cellby the presence of the protein that it encodes.

A “retroviral particle”, “transducing particle”, or “transductionparticle” refers to a replication-defective or replication-competentvirus capable of transducing non-viral DNA or RNA into a cell.

The terms “transformation”, “transduction” and “transfection” all denotethe introduction of a polynucleotide into an avian blastodermal cell.“Magnum” is that part of the oviduct between the infundibulum and theisthmus containing tubular gland cells that synthesize and secrete theegg white proteins of the egg.

A “MDOT promoter”, as used herein, is a synthetic promoter which isactive in the tubular gland cells of the magnum of the oviduct amongstother tissues. MDOT is comprised of elements from the ovomucoid (MD) andovotransferrin (TO) promoters (FIG. 13).

The term “optimized” is used in the context of “optimized codingsequence”, wherein the most frequently used codons for each particularamino acid found in the egg white proteins ovalbumin, lysozyme,ovomucoid, and ovotransferrin are used in the design of the optimizedhuman interferon-α 2b (IFN-α 2b) polynucleotide sequence that isinserted into vectors of the present invention. More specifically, theDNA sequence for optimized human IFN-α 2b is based on the hen oviductoptimized codon usage and is created using the BACKTRANSLATE program ofthe Wisconsin Package, Version 9.1 (Genetics Computer Group Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. For example, the percent usage for the four codons of theamino acid alanine in the four egg white proteins is 34% for GCU, 31%for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU is used as thecodon for the majority of alanines in the optimized human IFN-α 2bcoding sequence. The vectors containing the gene for optimized humanIFN-α 2b are used to produce transgenic avians that express transgenicpoultry derived IFN-α 2b (TPD IFN-α 2b) in their tissues and eggs.Similarly, the above method is employed for the design of other codingsequences proteins such as human erythropoietin (EPO) or other proteinswhich may be produced in accordance with the invention.

By the methods of the present invention, transgenes can be introducedinto avian embryonic blastodermal cells to produce a transgenic chicken,transgenic turkey, transgenic quail and other avian species, that carrythe transgene in the genetic material of its germ-line tissue. Theblastodermal cells are typically stage VII-XII cells, or the equivalentthereof, and in one embodiment are near stage X. The cells useful in thepresent invention include embryonic germ (EG) cells, embryonic stem (ES)cells & primordial germ cells (PGCs). The embryonic blastodermal cellsmay be isolated freshly, maintained in culture, or reside within anembryo.

The vectors useful in carrying out the methods of the present inventionare described herein. These vectors may be used for stable introductionof an exogenous coding sequence into the genome of an avian.Alternatively, the vectors may be used to produce exogenous proteins inspecific tissues of an avian, for example, in the oviduct tissue of anavian. The vectors may also be used in methods to produce avian eggswhich contain exogenous protein. In one embodiment, the coding sequenceand the promoter are both positioned between 5′ and 3′ LTRs beforeintroduction into blastodermal cells. In one embodiment, the vector isretroviral and the coding sequence and the promoter are both positionedbetween the 5′ and 3′ LTRs of the retroviral vector. In one usefulembodiment, the LTRs or retroviral vector is derived from the avianleukosis virus (ALV), murine leukemia virus (MLV), or lentivirus.

In one embodiment, the vector includes a signal peptide coding sequencewhich is operably linked to the coding sequence, so that upontranslation in a cell, the signal peptide will direct secretion of theexogenous protein expressed by the vector into the egg white of a hardshell egg. The vector may include a marker gene, wherein the marker geneis operably linked to a promoter.

In some cases, introduction of a vector of the present invention intothe embryonic blastodermal cells is performed with embryonicblastodermal cells that are either freshly isolated or in culture. Thetransgenic cells are then typically injected into the subgerminal cavitybeneath a recipient blastoderm in an egg. In some cases, however, thevector is delivered directly to the cells of a blastodermal embryo.

In one embodiment of the invention, vectors used for transfectingblastodermal cells and generating stable integration into the aviangenome contain a coding sequence and a promoter in operational andpositional relationship to express the coding sequence in the tubulargland cell of the magnum of the avian oviduct, wherein the codingsequence codes for an exogenous protein which is deposited in the eggwhite of a hard shell egg. The promoter may optionally be a segment ofthe ovalbumin promoter region which is sufficiently large to directexpression of the coding sequence in the tubular gland cells. Theinvention involves truncating the ovalbumin promoter and/or condensingthe critical regulatory elements of the ovalbumin promoter so that itretains sequences required for expression in the tubular gland cells ofthe magnum of the oviduct, while being small enough that it can bereadily incorporated into vectors. In one embodiment, a segment of theovalbumin promoter region may be used. This segment comprises the5′-flanking region of the ovalbumin gene. The total length of theovalbumin promoter segment may be from about 0.88 kb to about 7.4 kb inlength, and is preferably from about 0.88 kb to about 1.4 kb in length.The segment preferably includes both the steroid-dependent regulatoryelement and the negative regulatory element of the ovalbumin gene. Thesegment optionally also includes residues from the 5′ untranslatedregion (5′ UTR) of the ovalbumin gene. Hence, the promoter may bederived from the promoter regions of the ovalbumin-, lysozyme-,conalbumin-, ovomucoid-, ovotransferrin- or ovomucin genes (FIG. 14). Anexample of such a promoter is the synthetic MDOT promoter which iscomprised of elements from the ovomucoid and ovotransferrin promoter(FIG. 13). The promoter may also be a promoter that is largely, but notentirely, specific to the magnum, such as the lysozyme promoter. Thepromoter may also be a mouse mammary tumor virus (MMTV) promoter.Alternatively, the promoter may be a constitutive promoter (e.g., acytomegalovirus (CMV) promoter, a rous-sarcoma virus (RSV) promoter, amurine leukemia virus (MLV) promoter, etc.). In a preferred embodimentof the invention, the promoter is a cytomegalovirus (CMV) promoter, aMDOT promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemiavirus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, anovalbumin promoter, a lysozyme promoter, a conalbumin promoter, anovomucoid promoter, an ovomucin promoter, and an ovotransferrinpromoter. Optionally, the promoter may be at least one segment of apromoter region, such as a segment of the ovalbumin-, lysozyme-,conalbumin-, ovomucoid-, ovomucin-, and ovotransferrin promoter region.In one embodiment, the promoter is a CMV promoter.

FIGS. 1A and 1B illustrate examples of ovalbumin promoter expressionvectors. Gene X is a coding sequence which encodes an exogenous protein.Bent arrows indicate the transcriptional start sites. In one example,the vector contains 1.4 kb of the 5′ flanking region of the ovalbumingene (FIG. 1A). The sequence of the “−1.4 kb promoter” of FIG. 1Acorresponds to the sequence starting from approximately 1.4 kb upstream(1.4 kb) of the ovalbumin transcription start site and extendingapproximately 9 residues into the 5′ untranslated region of theovalbumin gene. The approximately 1.4 kb-long segment harbors twocritical regulatory elements, the steroid-dependent regulatory element(SDRE) and the negative regulatory element (NRE). The NRE is so namedbecause it contains several negative regulatory elements which block thegene's expression in the absence of hormones (e.g., estrogen). A shorter0.88 kb segment also contains both elements. In another example, thevector contains approximately 7.4 kb of the 5′ flanking region of theovalbumin gene and harbors two additional elements (HS-III and HS-IV),one of which is known to contain a functional region enabling inductionof the gene by estrogen (FIG. 1B). A shorter 6 kb segment also containsall four elements and could optionally be used in the present invention.

Each vector used for random integration according to the presentinvention preferably comprises at least one 1.2 kb element from thechicken β-globin locus which insulates the gene within from bothactivation and inactivation at the site of insertion into the genome. Inone embodiment, two insulator elements are added to one end of theovalbumin gene construct. In the β-globin locus, the insulator elementsserve to prevent the distal locus control region (LCR) from activatinggenes upstream from the globin gene domain, and have been shown toovercome position effects in transgenic flies, indicating that they canprotect against both positive and negative effects at the insertionsite. The insulator element(s) are only needed at either the 5′ or 3′end of the gene because the transgenes are integrated in multiple,tandem copies effectively creating a series of genes flanked by theinsulator of the neighboring transgene. In another embodiment, theinsulator element is not linked to the vector but is cotransfected withthe vector. In this case, the vector and the element are joined intandem in the cell by the process of random integration into the genome.

Each vector may optionally also comprise a marker gene to allowidentification and enrichment of cell clones which have stablyintegrated the expression vector. The expression of the marker gene isdriven by a ubiquitous promoter that drives high levels of expression ina variety of cell types. In one embodiment of the invention, the markergene is human interferon driven by a lysozyme promoter. In anotherembodiment the green fluorescent protein (GFP) reporter gene (Zolotukhinet al., J. Virol 70:4646-4654 (1995)) is driven by the Xenopuselongation factor 1-α (ef-1-α) promoter (Johnson and Krieg, Gene147:223-26 (1994)). The Xenopus ef-1-α promoter is a strong promoterexpressed in a variety of cell types. The GFP contains mutations thatenhance its fluorescence and is humanized, or modified such that thecodons match the codon usage profile of human genes. Since avian codonusage is virtually the same as human codon usage, the humanized form ofthe gene is also highly expressed in avian blastodermal cells. Inalternative embodiments, the marker gene is operably linked to one ofthe ubiquitous promoters of HSV tk, CMV, β-actin, or RSV.

While human and avian codon usage is well matched, where a nonvertebrategene is used as the coding sequence in the transgene, the nonvertebrategene sequence may be modified to change the appropriate codons such thatcodon usage is similar to that of humans and avians.

Transfection of the blastodermal cells may be mediated by any number ofmethods known to those of ordinary skill in the art. The introduction ofthe vector to the cell may be aided by first mixing the nucleic acidwith polylysine or cationic lipids which help facilitate passage acrossthe cell membrane. However, introduction of the vector into a cell ispreferably achieved through the use of a delivery vehicle such as aliposome or a virus. Viruses which may be used to introduce the vectorsof the present invention into a blastodermal cell include, but are notlimited to, retroviruses, adenoviruses, adeno-associated viruses, herpessimplex viruses, and vaccinia viruses.

In one method of transfecting blastodermal cells, a packagedretroviral-based vector is used to deliver the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome.

As an alternative to delivering retroviral transduction particles to theembryonic blastodermal cells in an embryo, helper cells which producethe retrovirus can be delivered to the blastoderm.

Useful retrovirus for randomly introducing a transgene into the aviangenome is the replication-deficient avian leucosis virus (ALV), thereplication-deficient murine leukemia virus (MLV), or the lentivirus. Inorder to produce an appropriate retroviral vector, a pNLB vector ismodified by inserting a region of the ovalbumin promoter and one or moreexogenous genes between the 5′ and 3′ long terminal repeats (LTRs) ofthe retrovirus genome. The invention contemplates that any codingsequence placed downstream of a promoter that is active in tubular glandcells will be expressed in the tubular gland cells. For example, theovalbumin promoter will be expressed in the tubular gland cells of theoviduct magnum because the ovalbumin promoter drives the expression ofthe ovalbumin protein and is active in the oviduct tubular gland cells.While a 7.4 kb ovalbumin promoter has been found to produce the mostactive construct when assayed in cultured oviduct tubular gland cells,the ovalbumin promoter is preferably shortened for use in the retroviralvector. In one embodiment, the retroviral vector comprises a 1.4 kbsegment of the ovalbumin promoter; a 0.88 kb segment would also suffice.

Any of the vectors of the present invention may also optionally includea coding sequence encoding a signal peptide that will direct secretionof the protein expressed by the vector's coding sequence from thetubular gland cells of the oviduct. This aspect of the inventioneffectively broadens the spectrum of exogenous proteins that may bedeposited in avian eggs using the methods of the invention. Where anexogenous protein would not otherwise be secreted, the vector containingthe coding sequence is modified to comprise a DNA sequence comprisingabout 60 bp encoding a signal peptide from the lysozyme gene. The DNAsequence encoding the signal peptide is inserted in the vector such thatit is located at the N-terminus of the protein encoded by the DNA.

FIGS. 2A-2D illustrate examples of suitable retroviral vectorconstructs. The vector construct is inserted into the avian genome with5′ and 3′ flanking LTRs. Neo is the neomycin phosphotransferase gene.Bent arrows indicate transcription start sites.

FIGS. 2A and 2B illustrate LTR and oviduct transcripts with a sequenceencoding the lysozyme signal peptide (LSP), whereas FIGS. 2C and 2Dillustrate transcripts without such a sequence. There are two parts tothe retroviral vector strategy. Any protein that contains a eukaryoticsignal peptide may be cloned into the vectors depicted in FIGS. 2B and2D. Any protein that is not ordinarily secreted may be cloned into thevectors illustrated in FIGS. 2A and 2B to allow for its secretion fromthe tubular gland cells.

FIG. 2E illustrates the strategy for cloning an exogenous gene into alysozyme signal peptide vector. The polymerase chain reaction is used toamplify a copy of a coding sequence, gene X, using a pair ofoligonucleotide primers containing restriction enzyme sites that enablethe insertion of the amplified gene into the plasmid after digestionwith the two enzymes. The 5′ and 3′ oligonucleotides contain the Bsu36Iand Xba1 restriction sites, respectively.

Another aspect of the invention involves the use of internal ribosomeentry site (IRES) elements in any of the vectors of the presentinvention to allow the translation of two or more proteins from adicistronic or polycistronic mRNA (Example 15). The IRES units are fusedto 5′ ends of one or more additional coding sequences which are theninserted into the vectors at the end of the original coding sequence, sothat the coding sequences are separated from one another by an IRES(FIGS. 2F, 15A and 15D). Pursuant to this aspect of the invention,post-translational modification of the product is facilitated becauseone coding sequence may encode an enzyme capable of modifying the othercoding sequence product. For example, the first coding sequence mayencode collagen which would be hydroxylated and made active by theenzyme encoded by the second coding sequence. In the retroviral vectorexample of FIG. 2F, an internal ribosome entry site (IRES) element ispositioned between two exogenous coding sequences (gene X and gene Y).The IRES allows both protein X and protein Y to be translated from thesame transcript the transcription of which is directed by a promotersuch as the ovalbumin promoter. Bent arrows indicate transcription startsites. The expression of the protein encoded by gene X is expected to behighest in tubular gland cells, where it is specifically expressed butnot secreted. The protein encoded by gene Y is also expressedspecifically in tubular gland cells but because it is efficientlysecreted, protein Y is packaged into the eggs. In the retroviral vectorexample of FIGS. 15A and 15D, the light chain (LC) and heavy chain (HC)of a human monoclonal antibody are expressed from a single vector,pCMV-LC-emcvIRES-HC, by placement of an IRES from theencephalomyocarditis virus (EMCV). Transcription is driven by a CMVpromoter. (See also Murakami et al. (1997) “High-level expression ofexogenous genes by replication-competent retrovirus vectors with aninternal ribosomal entry site” Gene 202:23-29; Chen et al. (1999)“Production and design of more effective avian replication-incompetentretroviral vectors” Dev. Biol. 214:370-384; Noel et al. (2000)“Sustained systemic delivery of monoclonal antibodies by geneticallymodified skin fibroblasts” J. Invest. Dermatol. 115:740-745).

In another aspect of the invention, the coding sequences of vectors usedin any of the methods of the present invention are provided with a 3′untranslated region (3′ UTR) to confer stability to the RNA produced.When a 3′ UTR is added to a retroviral vector, the orientation of thepromoter, gene X and the 3′ UTR must be reversed in the construct, sothat the addition of the 3′ UTR will not interfere with transcription ofthe full-length genomic RNA. In one embodiment, the 3′ UTR may be thatof the ovalbumin or lysozyme genes, or any 3′ UTR that is functional ina magnum cell, i.e., the SV40 late region.

In an alternative embodiment of the invention, a constitutive promoter(e.g., CMV) is used to express the coding sequence of a transgene in themagnum of an avian. In this case, expression is not limited to themagnum; expression also occurs in other tissues within the avian (e.g.,blood). The use of such a transgene, which includes a constitutivepromoter and a coding sequence, is particularly suitable for effectingor driving the expression of a protein in the oviduct and the subsequentsecretion of the protein into the egg white (see FIG. 8A for an exampleof a CMV driven construct, such as the pNLB-CMV-IFN vector forexpressing IFN-α 2b in chickens).

FIG. 3A shows a schematic of the replication-deficient avian leukosisvirus (ALV)-based vector pNLB, a vector which is suitable for use in theinvention. In the pNLB vector, most of the ALV genome is replaced by theneomycin resistance gene (Neo) and the lacZ gene, which encodesb-galactosidase. FIG. 3B shows the vector pNLB-CMV-BL, in which lacZ hasbeen replaced by the CMV promoter and the β-lactamase coding sequence(β-La or BL). Construction of the vector is reported in the specificexamples (Example 1, vide infra). β-lactamase is expressed from the CMVpromoter and utilizes a polyadenylation signal (pA) in the 3′ longterminal repeat (LTR). The β-Lactamase protein has a natural signalpeptide; thus, it is found in blood and in egg white.

Avian embryos are transduced with the pNLB-CMV-BL vector (Example 2,vide infra). The egg whites of eggs from the resulting stably transducedhens contain up to 60 micrograms (μg) of secreted, active β-lactamaseper egg (Examples 2 and 3, vide infra).

FIGS. 8A and 8B illustrates the pNLB-CMV-IFN vector used for expressinginterferon-α 2b (IFN-α 2b) and the pNLB-MDOT-EPO vector used forexpressing erythropoietin (EPO), respectively. Both exogenous proteins(EPO, IFN) are expressed in avians, preferably chicken and turkey.

The pNLB-MDOT-EPO vector is created by substituting an EPO encodingsequence for the BL encoding sequence (Example 10, vide infra). In oneembodiment, a synthetic promoter called MDOT is employed to driveexpression of EPO. MDOT contains elements from both the ovomucoid andovotransferrin promoter. The DNA sequence for human EPO is based on henoviduct optimized codon usage as created using the BACKTRANSLATE programof the Wisconsin Package, version 9.1 (Genetics Computer Group, Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. The EPO DNA sequence is synthesized and cloned into the vectorand the resulting plasmid is pNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2).In one embodiment, transducing particles (i.e., transduction particles)are produced for the vector, and these transducing particles are titeredto determine the appropriate concentration that can be used to injectembryos. Eggs are then injected with transducing particles after whichthey hatch about 21 days later.

The exogenous protein levels such as the EPO levels can then be measuredby an ELISA assay from serum samples collected from chicks one weekafter hatch. Male birds are selected for breeding, wherein birds arescreened for G0 roosters which contain the EPO transgene in their sperm.Preferably, roosters with the highest levels of the transgene in theirsperm samples are bred to nontransgenic hens by artificial insemination.Blood DNA samples are screened for the presence of the transgene. Anumber of chicks are usually found to be transgenic (G1 avians). Chickserum is tested for the presence of human EPO (e.g., ELISA assay). Theegg white in eggs from G1 hens is also tested for the presence of humanEPO. The EPO (i.e., derived from the optimized coding sequence of humanEPO) present in eggs of the present invention is biologically active(Example 11).

Similarly, the pNLB-CMV-IFN vector (FIG. 8A) is created by substitutingan IFN encoding sequence for the BL encoding sequence (Example 12, videinfra). In one embodiment, a constitutive cytomegalovirus (CMV) promoteris employed to drive expression of IFN. More specifically, the IFNcoding sequence is controlled by the cytomegalovirus (CMV) immediateearly promoter/enhancer and SV40 polyA site. FIG. 8A illustratespNLB-CMV-IFN used for expressing IFN in avians, for example, chicken andturkey. An optimized coding sequence is created for human IFN-α 2b,wherein the most frequently used codons for each particular amino acidfound in the egg white proteins ovalbumin, lysozyme, ovomucoid, andovotransferrin are used in the design of the human IFN-α 2b sequencethat is inserted into vectors of the present invention. Morespecifically, the DNA sequence for the optimized human IFN-α 2b (FIG.11A) is based on the hen oviduct optimized codon usage and is createdusing the BACKTRANSLATE program (supra) with a codon usage tablecompiled from the chicken (Gallus gallus) ovalbumin, lysozyme,ovomucoid, and ovotransferrin proteins. For example, the percent usagefor the four codons of the amino acid alanine in the four egg whiteproteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG.Therefore, GCU is used as the codon for the majority of alanines in theoptimized human IFN-α 2b sequence. The vectors containing the gene forthe optimized human IFN-α 2b sequence are used to create transgenicavians that express TPD IFN-α 2b in their tissues and eggs.

Transducing particles (i.e., transduction particles) are produced forthe vector and titered to determine the appropriate concentration thatcan be used to inject embryos (Example 2, vide infra). Thus, chimericavians are produced (see also Example 13, vide infra). Avian eggs arewindowed according to the Speksnijder procedure (U.S. Pat. No.5,897,998), and eggs are injected with transducing particles Eggs hatchabout 21 days after injection. hIFN levels are measured (e.g., ELISAassay) from serum samples collected from chicks one week after hatch. Aswith EPO (supra), male birds are selected for breeding. In order toscreen for G0 roosters which contain the IFN transgene in their sperm,DNA is extracted from rooster sperm samples. The G0 roosters with thehighest levels of the transgene in their sperm samples are bred tonontransgenic hens by artificial insemination. Blood DNA samples arescreened for the presence of the transgene. The serum of transgenicroosters is tested for the presence of hIFN (e.g., ELISA assay). If theexogenous protein is confirmed the sperm of the transgenic roosters isused for artificial insemination of nontransgenic hens. A certainpercent of the offspring will then contain the transgene (e.g., morethan 50%). When IFN (i.e., derived from the optimized coding sequence ofhuman IFN) is present in eggs of the present invention, the IFN may betested for biological activity. As with EPO, such eggs usually containbiologically active IFN, such as TPD IFN-α 2b (FIG. 11B).

The methods of the invention which provide for the production ofexogenous protein in the avian oviduct and the production of eggs whichcontain exogenous protein involve an additional step subsequent toproviding a suitable vector and introducing the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome. The subsequent step involves deriving a mature transgenic avianfrom the transgenic blastodermal cells produced in the previous steps.Deriving a mature transgenic avian from the blastodermal cellsoptionally involves transferring the transgenic blastodermal cells to anembryo and allowing that embryo to develop fully, so that the cellsbecome incorporated into the avian as the embryo is allowed to develop.The resulting chick is then grown to maturity. In one embodiment, thecells of a blastodermal embryo are transfected or transduced with thevector directly within the embryo (Example 2). The resulting embryo isallowed to develop and the chick allowed to mature.

In either case, the transgenic avian so produced from the transgenicblastodermal cells is known as a founder. Some founders will carry thetransgene in the tubular gland cells in the magnum of their oviducts.These avians will express the exogenous protein encoded by the transgenein their oviducts. The exogenous protein may also be expressed in othertissues (e.g., blood) in addition to the oviduct. If the exogenousprotein contains the appropriate signal sequence(s), it will be secretedinto the lumen of the oviduct and into the egg white of the egg. Somefounders are germ-line founders (Examples 8 and 9). A germ-line founderis a founder that carries the transgene in genetic material of itsgerm-line tissue, and may also carry the transgene in oviduct magnumtubular gland cells that express the exogenous protein. Therefore, inaccordance with the invention, the transgenic avian will have tubulargland cells expressing the exogenous protein, and the offspring of thetransgenic avian will also have oviduct magnum tubular gland cells thatexpress the exogenous protein. Alternatively, the offspring express aphenotype determined by expression of the exogenous gene in specifictissue(s) of the avian (Example 6, Table 2). In one embodiment of theinvention, the transgenic avian is a chicken or a turkey.

The invention can be used to express, in large yields and at low cost,desired proteins including those used as human and animalpharmaceuticals, diagnostics, and livestock feed additives. For example,the invention includes transgenic avians that produce such proteins andeggs laid by the transgenic avians which contain the protein, forexample, in the egg white. The present invention is contemplated for usein the production of any desired protein including pharmaceuticalproteins with the requisite that the coding sequence of the protein canbe introduced into an oviduct cell in accordance with the presentinvention. In fact, all proteins tested thus far for heterologousproduction in accordance with the present invention, includinginterferon α 2b, GM-CSF, interferon β, erythropoietin, G-CSF, CTLA4-Fcfusion protein and β-lactamase, have been produced successfullyemploying the methods disclosed herein.

The production of human proteins as disclosed herein is of particularinterest. The human form of each of the proteins disclosed herein forwhich there is a human form, is contemplated for production inaccordance with the invention.

Proteins contemplated for production as disclosed herein include, butare not limited to, fusion proteins, growth hormones, cytokines,structural proteins and enzymes including human growth hormone,interferon, lysozyme, and β-casein, albumin, α-1 antitrypsin,antithrombin III, collagen, factors VIII, IX, X (and the like),fibrinogen, insulin, lactoferrin, protein C, erythropoietin (EPO),granulocyte colony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), tissue-type plasminogen activator(tPA), somatotropin, and chymotrypsin. Modified immunoglobulins andantibodies, including immunotoxins which bind to surface antigens onhuman tumor cells and destroy them, can also be produced as disclosedherein.

Other specific examples of therapeutic proteins which may be produced asdisclosed herein include, without limitation, factor VIII, b-domaindeleted factor VIII, factor VIIa, factor IX, anticoagulants; hirudin,alteplase, tpa, reteplase, tpa, tpa-3 of 5 domains deleted, insulin,insulin lispro, insulin aspart, insulin glargine, long-acting insulinanalogs, hgh, glucagons, tsh, follitropin-beta, fsh, gm-csf, pdgh, ifnalpha2, ifn alpha2a, ifn alpha2b, inf-apha, inf-beta 1b, ifn-beta 1a,ifn-gamma1b, il-2, il-11, hbsag, ospa, murine mab directed againstt-lymphocyte antigen, murine mab directed against tag-72,tumor-associated glycoprotein, fab fragments derived from chimeric mabdirected against platelet surface receptor gpII(b)/III(a), murine mabfragment directed against tumor-associated antigen ca125, murine mabfragment directed against human carcinoembryonic antigen, cea, murinemab fragment directed against human cardiac myosin, murine mab fragmentdirected against tumor surface antigen psma, murine mab fragments(fab/fab2 mix) directed against hmw-maa, murine mab fragment (fab)directed against carcinoma-associated antigen, mab fragments (fab)directed against nca 90, a surface granulocyte nonspecific crossreacting antigen, chimeric mab directed against cd20 antigen found onsurface of b lymphocytes, humanized mab directed against the alpha chainof the il2 receptor, chimeric mab directed against the alpha chain ofthe il2 receptor, chimeric mab directed against tnf-alpha, humanized mabdirected against an epitope on the surface of respiratory synctialvirus, humanized mab directed against her 2, human epidermal growthfactor receptor 2, human mab directed against cytokeratintumor-associated antigen anti-ctla4, chimeric mab directed against cd 20surface antigen of b lymphocytes dornase-alpha dnase, betaglucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusion protein,tnfr-lgg fragment fusion protein laronidase, dnaases, alefacept,darbepoetin alfa (colony stimulating factor), tositumomab, murine mab,alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroidhormone derivatives, adalimumab (lgg1), anakinra, biological modifier,nesiritide, human b-type natriuretic peptide (hbnp), colony stimulatingfactors, pegvisomant, human growth hormone receptor antagonist,recombinant activated protein c, omalizumab, immunoglobulin e (lge)blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin,somatotropin, thymosin, parathyroid hormone, pigmentary hormones,somatomedin, erythropoietin, luteinizing hormone, chorionicgonadotropin, hypothalmic releasing factors, etanercept, antidiuretichormones, prolactin and thyroid stimulating hormone.

The invention includes methods for producing multimeric proteinsincluding immunoglobulins, such as antibodies, and antigen bindingfragments thereof. Thus, in one embodiment of the present invention, themultimeric protein is an immunoglobulin, wherein the first and secondheterologous polypeptides are immunoglobulin heavy and light chainsrespectively.

In certain embodiments, an immunoglobulin polypeptide encoded by thetranscriptional unit of at least one expression vector may be animmunoglobulin heavy chain polypeptide comprising a variable region or avariant thereof, and may further comprise a D region, a J region, a Cregion, or a combination thereof. An immunoglobulin polypeptide encodedby an expression vector may also be an immunoglobulin light chainpolypeptide comprising a variable region or a variant thereof, and mayfurther comprise a J region and a C region. The present invention alsocontemplates multiple immunoglobulin regions that are derived from thesame animal species, or a mixture of species including, but not only,human, mouse, rat, rabbit and chicken. In certain embodiments, theantibodies are human or humanized.

In other embodiments, the immunoglobulin polypeptide encoded by at leastone expression vector comprises an immunoglobulin heavy chain variableregion, an immunoglobulin light chain variable region, and a linkerpeptide thereby forming a single-chain antibody capable of selectivelybinding an antigen.

Examples of therapeutic antibodies that may be produced in methods ofthe invention include, but are not limited, to HERCEPTIN™ (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REOPRO™(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX™(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX™ which is amurine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFRIgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3integrin antibody (Applied Molecular Evolution/MedImmune); Campath;Campath 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody(Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody(Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG1antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is ahumanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanizedanti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate anti-CD80antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelled murineanti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanizedanti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (CS) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CATIBASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A isa humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is ahumanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanizedanti-VLA-4 IgG antibody (Elan); CAT-152, a human anti-TGF-β₂ antibody(Cambridge Ab Tech); Cetuximab (BMS) is a monoclonal anti-EGF receptor(EGFr) antibody; Bevacizuma (Genentech) is an anti-VEGF human monoclonalantibody; Infliximab (Centocore, JJ) is a chimeric (mouse and human)monoclonal antibody used to treat autoimmune disorders; Gemtuzumabozogamicin (Wyeth) is a monoclonal antibody used for chemotherapy; andRanibizumab (Genentech) is a chimeric (mouse and human) monoclonalantibody used to treat macular degeneration.

In one aspect, the invention is drawn to G-CSF produced in poultry oravains. In one aspect, the invention is drawn to G-CSF with a poultryderived glycosylation pattern (TPD G-CSF) wherein the G-CSF is obtainedfrom avian cells, for example, avian cells of a chicken, quail orturkey. Also included in the invention are the human proteins includingcytokines such as G-CSF produced in poultry in isolated or purified formand human proteins including cytokines such as G-CSF produced in poultrypresent in pharmaceutical compositions. The isolation of the proteinsincluding G-CSF can be accomplished by methodologies readily apparent toa practitioner skilled in the art of protein purification. The make-upof formulations useful for producing pharmaceutical compositions arealso well known in the art.

The present invention encompasses transgenic poultry derived therapeuticor pharmaceutical proteins having a poultry derived glycosylationpattern which are derived from avians. For example, the inventionincludes interferon-α 2 (TPD IFN-α 2) derived from avians. TPD IFN-α 2(e.g., species type b) exhibits a new glycosylation pattern and containsnew glyco forms (bands 4 and 5 are α-Gal extended disaccharides; seeFIG. 9) not normally seen in human peripheral blood leukocyte derivedinterferon-α 2 (PBL IFN-α 2b). TPD IFN-α 2b also contains O-linkedcarbohydrate structures that are similar to human PBL IFN-α 2b and ismore efficiently produced in chickens than the human form.

The present invention contemplates an isolated polynucleotide comprisingthe optimized polynucleotide sequence of proteins produced as disclosedherein. For example, the invention includes avian optimized codingsequence for human IFN-α 2b, i.e., recombinant transgenic poultryderived interferon-α 2b (TPD IFN-α 2b) (SEQ ID NO: 1). The codingsequence for optimized human IFN-α 2b includes 498 nucleic acids and 165amino acids (see SEQ ID NO: 1 and FIG. 11A). Similarly, the codingsequence for natural human IFN-α 2b includes 498 nucleotides (NCBIAccession Number AF405539 and GI:15487989) and 165 amino acids (NCBIAccession Number AAL01040 and GI:15487990). The most frequently usedcodons for each particular amino acid found in the egg white proteinsovalbumin, lysozyme, ovomucoid, and ovotransferrin are used in thedesign of the optimized human IFN-α 2b coding sequence which is insertedinto vectors of the present invention. More specifically, the DNAsequence for the optimized human IFN-α 2b is based on the hen oviductoptimized codon usage and is created using the BACKTRANSLATE program ofthe Wisconsin Package, Version 9.1 (Genetics Computer Group Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. For example, the percent usage for the four codons of theamino acid alanine in the four egg white proteins is 34% for GCU, 31%for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU is used as thecodon for the majority of alanines in the optimized human IFN-α 2bcoding sequence. The vectors containing the gene for optimized humanIFN-α 2b are used to create transgenic avians that express TPD IFN-α 2bin their tissues and eggs.

As discussed in Example 13 (vide infra), TPD IFN-α 2b is produced inchicken. However, TPD IFN-α 2b may also be produced in turkey and otheravian species such as quail. In a preferred embodiment of the invention,TPD IFN-α 2b is expressed in chicken and turkey and their hard shelleggs. A carbohydrate analysis (Example 14, vide infra), including amonosaccharide analysis and FACE analysis, reveals the sugar make-up ornovel glycosylation pattern of the protein. As such, TPD IFN-α 2b showsthe following monosaccharide residues: N-Acetyl-Galactosamine (NAcGal),Galactose (Gal), N-Acetyl-Glucosamine (NAcGlu), and Sialic acid (SA).However, there is no N-linked glycosylation in TPD IFN-α2b. Instead, TPDIFN-α 2b is O-glycosylated at Thr-106. This type of glycosylation issimilar to human IFN-α 2, wherein the Thr residue at position 106 isunique to IFN-α 2. Similar to natural IFN-α, TPD IFN-α 2b does not havemannose residues. A FACE analysis reveals 6 bands (FIG. 9) thatrepresent various sugar residues, wherein bands 1, 2 and 3 areun-sialylated, mono-sialylated, and di-sialylated, respectively (FIG.10). The sialic acid (SA) linkage is alpha 2-3 to Galactose (Gal) andalpha 2-6 to N-Acetyl-Galactosamine (NAcGal). Band 6 represents anun-sialylated tetrasaccharide. Bands 4 and 5 are alpha-Galactose(alpha-Gal) extended disaccharides that are not seen in human PBL IFN-α2b or natural human IFN (natural hIFN). FIG. 10 shows the comparison ofTPD IFN-α 2b (egg white hIFN) and human PBL IFN-α 2b (natural hIFN).Minor bands are present between bands 3 and 4 and between bands 4 and 5in TPD IFN-α 2b (vide infra).

The present invention contemplates an isolated polypeptide sequence (SEQID NO: 2) of TPD IFN-α 2b (see also FIG. 11B) and a pharmaceuticalcomposition thereof, wherein the protein is O-glycosylated at Thr-106with one or more of the carbohydrate structures disclosed herein asfollows:

wherein Gal=Galactose,

-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.    In a one embodiment of the present invention, the percentages are as    follows:

wherein Gal=Galactose,

-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.

Minor bands are present between bands 3 and 4 and between bands 4 and 5which account for about 17% in TPD IFN-α 2b.

In one embodiment, the invention is directed to human proteins having apoultry derived glycosylation pattern. In one embodiment, the poultryderived glycosylation pattern is obtained from avian oviduct cells, forexample, tubular gland cells. For example, glycosylation patterns aredisclosed herein which have been demonstrated to be present on humanproteins produced in oviduct cells of a chicken in accordance with thepresent invention.

In one embodiment, the invention is directed to human G-CSF produced inavians (e.g., avian oviduct cells) such as chickens, turkey and quailhaving a poultry derived glycosylation pattern. The mature hG-CSF aminoacid sequence is shown in FIG. 18 C. Nucleotide sequence used herein toproduce G-CSF is shown in FIG. 18 A and in NCBI Accession NM 172219.Nucleotide sequences optimized for avian (e.g., chicken) codon usage arealso contemplated for use to produce G-CSF and other proteins such ashuman proteins produced in accordance with the invention.

The invention includes the eggs and the avians (e.g., chicken, turkeyand quail) that lay the eggs containing G-CSF molecules of the inventioncomprising one or more of the glycosylation structures shown below:

wherein Gal=Galactose,

-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.

In one embodiment, the invention includes a mixture of G-CSF moleculeswherein the mixture contains G-CSF molecules having a glycosylationstructure selected from one or more of Structure A, Structure B,Structure C, Structure D, Structure E, Structure F and Structure G. Theinvention also includes a mixture of G-CSF molecules wherein the mixturecontains G-CSF molecules having a glycosylation structure selected fromone or more of Structure A, Structure B, Structure C, Structure D,Structure E, Structure F and Structure G wherein the mixture is isolatedor purified, for example, purified from an egg or from egg whiteproduced in accordance with the invention. Also included is a mixture ofG-CSF molecules wherein the mixture contains G-CSF molecules having two,three, four, five or six of the structures: Structure A, Structure B,Structure C, Structure D, Structure E, Structure F and/or Structure G.Also included is a mixture of G-CSF molecules wherein the mixturecontains G-CSF molecules having two, three, four, five or six of thestructures: Structure A, Structure B. Structure C, Structure D,Structure E, Structure F and/or Structure G, that has been isolated orpurified, for example, purified from an egg or from egg white producedin accordance with the invention.

The invention also includes an individual G-CSF molecule comprising aStructure A. The invention also includes an individual G-CSF moleculecomprising a Structure B. The invention also includes an individualG-CSF molecule comprising a Structure C. The invention also includes anindividual G-CSF molecule comprising a Structure D. The invention alsoincludes an individual G-CSF molecule comprising a Structure E. Theinvention also includes an individual G-CSF molecule comprising aStructure F. The invention also includes an individual G-CSF moleculecomprising a Structure G. In one embodiment, the individual G-CSFmolecule is present in a mixture of G-CSF molecules that may be anisolated or purified mixture of G-CSF molecules, for example, themixture being purified from an egg or from egg white produced inaccordance with the invention. In one embodiment, the individual G-CSFmolecule is isolated or purified, for example, purified as disclosedherein (e.g., by HPLC as disclosed in Example 20).

The embodiments of the invention as specified herein regarding G-CSF,for example, mixtures of G-CSF molecules and individual G-CSF molecules(in the preceding two paragraphs), are also applicable in general foreach of the other proteins produced in accordance with the invention andtheir corresponding poultry derived glycosylation structures.

Transgenic chickens which lay eggs containing EPO were produced asdisclosed in Examples 22 and 23. In addition, a second line of EPOproducing chickens was produced essentially as described in examples 22and 23 except that a different producer cell line was used, as describedin US patent publication No. 2007/0077650, published May 5, 2007, thedisclosure of which is incorporated in its entirety herein by reference.This second line of EPO producing chickens appeared to have a deletionin the CMV promoter and in an LTR providing for an enhanced level ofproduction of EPO in the egg white of the resulting transgenic chickensas disclosed in U.S. patent application Ser. No. 11/880,838, filed Jul.24, 2007, the disclosure of which is incorporated in its entirety hereinby reference. This higher EPO producing line was used to obtain the EPOused for oligosaccharide analysis.

Proteins produced in transgenic avians in accordance with the inventioncan be purified from egg white by any useful procedure such as thoseapparent to a practitioner of ordinary skill in the art of proteinpurification. For example, the EPO produced in transgenic avians inaccordance with the invention can be purified from egg white by methodsapparent to practitioners of ordinary skill in the art of proteinpurification. An example of a purification protocol for EPO present inegg white is described in Example 24.

The human erythropoietin (hEPO) produced in chickens has been shown tocontain an O-linked carbohydrate chain and three N-linked carbohydratechains. The O-linked glycosylation has been shown to be at Ser-126 ofthe EPO and the N-linked glycosylations have been shown to be at Asn-24,Asn-38 and Asn-83. The mature erythropoietin amino acid sequenceproduced in accordance with the invention is shown in FIG. 19B. Thehuman nucleotide sequence encoding the EPO is shown in FIG. 19A.Nucleotide sequences optimized for avian (e.g., chicken) codon usage arealso contemplated for use to produce EPO and other proteins inaccordance with the invention.

Representative glycosylation structures have been determined for theerythropoietin of the invention and are shown in Example 25 and in FIGS.20 and 21. In particular, B-n, D-n, F-n, H-n, J-n, L-n, N-n, O-n, P-n,and Q-n have been identified as being present on the avian derived EPO.Also, evidence shows that one or more of oligosaccharide structures A-n,C-n, E-n, G-n, I-n, K-n and M-n may also be present on the EPO. Inaddition, data has indicated that there may be a second form of Q-n inwhich only four of the five terminal NAcGlu residues are present. Thissecond form of Q-n may be a precursor form of Q-n.

The invention includes the eggs and egg white and the avians (e.g.,chicken turkey and quail) that lay the eggs and produce the egg whitecontaining erythropoietin molecules of the invention comprising one ormore of the glycosylation structures disclosed herein.

In one embodiment, the invention includes a mixture of erythropoietinmolecules wherein the mixture contains erythropoietin molecules (e.g.,one or more erythropoietin molecules) having an O-linked glycosylationstructure selected from Structure A-o, Structure B-o and Structure C-o.Though O-linked glycosylation analysis to date have confirmed thepresence of A-o, B-o and C-o; Structure D-o, Structure E-o, StructureF-o and Structure G-o are also contemplated as being present on thepoultry derived human EPO. It has been determined that the primaryO-linked oligosaccharide present on the avian derived EPO appears to beC-o.

The invention also includes EPO having N-linked glycosylation structuresat three sites wherein the structures at each of the three sites areselected from one of A-n, B-n, C-n, D-n, E-n, F-n, G-n, H-n, I-n, J-n,K-n, L-n, M-n, N-n, O-n, P-n and Q-n.

The invention also includes a mixture of erythropoietin molecules (e.g.,more than one erythropoietin molecule) wherein some or all of theerythropoietin molecules have one or more glycosylation structuresselected from Structure A-o, Structure B-o, Structure C-o, StructureA-n, Structure B-n, Structure C-n, Structure D-n, Structure E-n,Structure F-n, Structure G-n, Structure H-n, Structure I-n, StructureJ-n, Structure K-n, Structure L-n, Structure M-n, Structure N-n,Structure O-n, Structure P-n, Structure Q-n. In one embodiment, themixture of erythropoietin molecules is purified or isolated, forexample, isolated from an egg or purified or isolated from egg whiteproduced in a transgenic avian.

The invention also includes an individual erythropoietin moleculecomprising a Structure A-o. The invention also includes an individualerythropoietin molecule comprising a Structure B-o. The invention alsoincludes an individual erythropoietin molecule comprising a StructureC-o. The invention also includes an individual erythropoietin moleculecomprising a Structure A-n. The invention also includes an individualerythropoietin molecule comprising a Structure B-n. The invention alsoincludes an individual erythropoietin molecule comprising a StructureC-n. The invention also includes an individual erythropoietin moleculecomprising a Structure D-n. The invention also includes an individualerythropoietin molecule comprising a Structure E-n. The invention alsoincludes an individual erythropoietin molecule comprising a StructureF-n. The invention also includes an individual erythropoietin moleculecomprising a Structure G-n. The invention also includes an individualerythropoietin molecule comprising a Structure H-n. The invention alsoincludes an individual erythropoietin molecule comprising a StructureI-n. The invention also includes an individual erythropoietin moleculecomprising a Structure J-n. The invention also includes an individualerythropoietin molecule comprising a Structure K-n. The invention alsoincludes an individual erythropoietin molecule comprising a StructureL-n. The invention also includes an individual erythropoietin moleculecomprising a Structure M-n. The invention also includes an individualerythropoietin molecule comprising a Structure N-n. The invention alsoincludes an individual erythropoietin molecule comprising a StructureN-n. The invention also includes an individual erythropoietin moleculecomprising a Structure P-n. The invention also includes an individualerythropoietin molecule comprising a Structure Q-n. In one embodiment,the individual erythropoietin molecule is present in a mixture oferythropoietin molecules which has been produced in a transgenic avian,e.g., a transgenic chicken. In one embodiment, the individualerythropoietin molecule is present in a mixture of erythropoietinmolecules which has been isolated or purified, for example, the mixtureis isolated or purified from an egg or from egg white produced by atransgenic avian. In one embodiment, the individual erythropoietinmolecule is isolated or purified.

The invention includes exemplary EPO molecules where each of the Asn-24,Asn-38 and Asn-83 glycosylation sites are glycosylated with one of theA-n, B-n, C-n, D-n, E-n, F-n, G-n, H-n, I-n, J-n, K-n, L-n, M-n, N-n,O-n, P-n and Q-n Structures and where and the Ser-126 is glycosylatedwith A-o, B-o or C-o.

MALDI-TOF-MS analysis of peptide products yielded from proteolyticdigests of the avian derived EPO of the invention have shown thatessentially the same oligosaccharide structures are present at each ofthe three N-linked glycosylation sites. That is, it appears that aboutthe same ratio of each of the N-linked oligosaccharides is present ateach of the three N-linked glycosylation sites on the EPO molecules.This indicates that other N-glycosylated exogenous proteins produced inaccordance with the invention may have similar N-linked glycosylationpatterns. In addition, data has shown that each of the three N-linkedsites is extensively glycosylated, each site being glycosylated greaterthan 95% of the time and possibly greater than 98% of the time, forexample, greater than 99% of the time. The erythropoietin analyzed wasproduced in a transgenic chicken which contained a transgene encodingthe amino acid sequence of the human 165 amino acid protein, aftercleavage of the signal peptide. However, it is expected that EPOproduced in a transgenic chicken using a nucleotide sequence encodingthe 166 amino acid form of EPO would result in the same complement ofoligosaccharides on the 166 amino acid protein as is found on the 165amino acid protein.

N-linked oligosaccharides attached to human EPO produced in transgenicchickens have a paucity of terminal sialic acid moieties. That is, onlyminor amounts of the N-linked oligosaccharide structures are terminallysialylated. This is in contrast to EPO produced in human cells and humanEPO produced in CHO cells where the N-linked oligosaccharide structuresare extensively terminally sialylated. In addition, terminal N-AcetylGlucosamine (NAcGlu) is present extensively on the N-linkedoligosaccharide structures of the EPO produced in transgenic chickenswhich is not the case for EPO produced in human cells and human EPOproduced in CHO cells. Further, fucose is not present on the N-linkedoligosaccharide structures of the EPO produced in transgenic chickens.However, fucose appears to be present on all or most N-linkedoligosaccharide structures of EPO produced in human cells and human EPOproduced in CHO cells.

Combinations of glycosylation structures are contemplated as beingattached to erythropoietin. For example, a human erythropoietin moleculemay be glycosylated with, A-o, A-n, B-n and C-n, or A-o, B-n, C-n andD-n, or A-o, D-n, E-n and F-n, or A-o, E-n, F-n and G-n, or B-o, A-n,D-n and H-n, or B-o, E-n, F-n and G-n, or B-o, A-n, A-n and A-n, or C-o,D-n, D-n and C-n, or C-o, F-n, G-n and H-n, or C-o, A-n, B-n and C-n, orC-o, A-n, B-n and H-n, or C-o, A-n, B-n and E-n, or C-o, A-n, B-n andH-n or other such combinations.

It is understood that though the reported method of making compositionsof the invention is in avians, the compositions are not limited thereto.For example, certain of the glycosylated protein molecules of theinvention may be produced in other organisms such as transgenic fish,transgenic mammals, for example, transgenic goats or in transgenicplants, such as tobacco and duck weed (Lemna minor).

It is also contemplated that the glycosylation structures demonstratedto be present on one protein of the invention may be present on anotherprotein of the invention. For example, glycosylation structures shown tobe present on TPD G-CSF may also be present on TPD GM-CSF, TPD IFNand/or other TPD proteins. In another example, it is contemplated thatthe glycosylation structures determined to be present on TPD IFN α2 maybe present on TPD G-CSF, TPD GM-CSF and/or other transgenic poultryderived (TPD) proteins. The invention also specifically contemplateshuman proteins in general having one or more of the TPD glycosyaltionstructures disclosed herein.

The invention also contemplates that pegylating proteins produced asdisclosed herein may be advantageous as discussed, for example, in U.S.patent application Ser. No. 11/584,832, filed Oct. 23, 2006, thedisclosure of which is incorporated it its entirety herein by reference.

While it is possible that, for use in therapy, therapeutic proteinsproduced in accordance with this invention may be administered in rawform, it is preferable to administer the therapeutic proteins as part ofa pharmaceutical formulation.

The invention thus further provides pharmaceutical formulationscomprising poultry derived glycosylated therapeutic proteins or apharmaceutically acceptable derivative thereof together with one or morepharmaceutically acceptable carriers thereof and, optionally, othertherapeutic and/or prophylactic ingredients and methods of administeringsuch pharmaceutical formulations. The carrier(s) must be “acceptable” inthe sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof. Methods oftreating a patient (e.g., quantity of pharmaceutical proteinadministered, frequency of administration and duration of treatmentperiod) using pharmaceutical compositions of the invention can bedetermined using standard methodologies known to physicians of skill inthe art.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal orparenteral. The pharmaceutical formulations include those suitable foradministration by injection including intramuscular, sub-cutaneous andintravenous administration. The pharmaceutical formulations also includethose for administration by inhalation or insufflation. The formulationsmay, where appropriate, be conveniently presented in discrete dosageunits and may be prepared by any of the methods well known in the art ofpharmacy. The methods of producing the pharmaceutical formulationstypically include the step of bringing the therapeutic proteins intoassociation with liquid carriers or finely divided solid carriers orboth and then, if necessary, shaping the product into the desiredformulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution; as a suspension; or as anemulsion. The active ingredient may also be presented as a bolus,electuary or paste. Tablets and capsules for oral administration maycontain conventional excipients such as binding agents, fillers,lubricants, disintegrants, or wetting agents. The tablets may be coatedaccording to methods well known in the art. Oral liquid preparations maybe in the form of, for example, aqueous or oily suspensions, solutions,emulsions, syrups or elixirs, or may be presented as a dry product forconstitution with water or other suitable vehicle before use. Suchliquid preparations may contain conventional additives such assuspending agents, emulsifying agents, non-aqueous vehicles (which mayinclude edible oils) or preservatives.

Therapeutic proteins of the invention may also be formulated forparenteral administration (e.g., by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The therapeutic proteins may beinjected by, for example, subcutaneous injections, intramuscularinjections, and intravenous infusions or injections.

The therapeutic proteins may take such forms as suspensions, solutions,or emulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. It isalso contemplated that the therapeutic proteins may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g., sterile,pyrogen-free water, before use.

For topical administration to the epidermis, the therapeutic proteinsproduced according to the invention may be formulated as ointments,creams or lotions, or as a transdermal patch. Ointments and creams may,for example, be formulated with an aqueous or oily base with theaddition of suitable thickening and/or gelling agents. Lotions may beformulated with an aqueous or oily base and will in general also containone or more emulsifying agents, stabilizing agents, dispersing agents,suspending agents, thickening agents or coloring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavored base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin or sucrose andacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably represented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by a mixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping in molds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

For intra-nasal administration the therapeutic proteins of the inventionmay be used as a liquid spray or dispersible powder or in the form ofdrops.

Drops may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents orsuspending agents. Liquid sprays are conveniently delivered frompressurized packs.

For administration by inhalation, therapeutic proteins according to theinvention may be conveniently delivered from an insufflator, nebulizeror a pressurized pack or other convenient means of delivering an aerosolspray. Pressurized packs may comprise a suitable propellant such asdichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount.

For administration by inhalation or insufflation, the therapeuticproteins according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form in, for example, capsules or cartridgesor, e.g., gelatin or blister packs from which the powder may beadministered with the aid of an inhalator or insufflator.

When desired, the above described formulations adapted to give sustainedrelease of the active ingredient, may be employed.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as antimicrobial agents, orpreservatives.

In a specific example, human EPO produced as disclosed herein, and whichmay be pegylated, is employed in a pharmaceutical formulation whereineach 1 mL contains 0.05 mg polysorbate 80, and is formulated at pH6.2±0.2 with 2.12 mg sodium phosphate monobasic monohydrate, 0.66 mgsodium phosphate dibasic anhydrous, and 8.18 mg sodium chloride in waterfor injection. In another specific example, human interferon alphaproduced as disclosed herein is employed in a pharmaceutical formulationcontaining 7.5 mg/ml sodium chloride, 1.8 mg/ml sodium phosphatedibasic, 1.3 mg/ml sodium phosphate monobasic, 0.1 mg/ml edetatedisodium dihydrate, 0.7 mg/ml Tween® 80 and 1.5 mg/ml m-cresol. Inanother specific example, human G-CSF produced as disclosed herein isemployed in a pharmaceutical formulation containing 0.82 mg/ml sodiumacetate, 2.8 μl/ml glacial acetic acid, 50 mg/ml mannitol and 0.04 mg/mlTween® 80.

In addition, it is contemplated that the therapeutic proteins of theinvention may be used in combination with other therapeutic agents.

Compositions or compounds of the invention can be used to treat avariety of conditions. For example, there are many conditions for whichtreatment therapies are known to practitioners of skill in the art inwhich therapeutic proteins obtained from cell culture (e.g., CHO cells)are employed. The present invention contemplates that the therapeuticproteins produced in an avian system containing a poultry derivedglycosyation pattern can be employed to treat such conditions. That is,the invention contemplates the treatment of conditions known to betreatable by conventionally produced therapeutic proteins by usingtherapeutic proteins produced in accordance with the invention. Forexample, erythropoietin produced in accordance with the invention can beused to treat human conditions such as anemia and kidney disease, e.g.,chronic renal failure (or other conditions which may be treatable byadministering EPO of the invention) and G-CSF produced in accordancewith the invention can be used to treat cancer patients, as understoodin the art.

Generally, the dosage administered will vary depending upon knownfactors such as age, health and weight of the recipient, type ofconcurrent treatment, frequency of treatment, and the like. Usually, adosage of active ingredient can be between about 0.0001 and about 10milligrams per kilogram of body weight. Precise dosage, frequency ofadministration and time span of treatment can be determined by aphysician skilled in the art of administration of the respectivetherapeutic protein.

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

EXAMPLE 1 Vector Construction

The lacZ gene of pNLB, a replication-deficient avian leukosis virus(ALV)-based vector (Cosset et al., 1991), was replaced with anexpression cassette consisting of a cytomegalovirus (CMV) promoter andthe reporter gene, β-lactamase. The pNLB and pNLB-CMV-BL vectorconstructs are diagrammed in FIGS. 3A and 3B, respectively.

To efficiently replace the lacZ gene of pNLB with a transgene, anintermediate adaptor plasmid was first created, pNLB-Adapter.pNLB-Adapter was created by inserting the chewed back ApaI/ApaI fragmentof pNLB (Cosset et al., J. Virol. 65:3388-94 (1991)) (in pNLB, the 5′ApaI resides 289 bp upstream of lacZ and the 3′ApaI resides 3′ of the 3′LTR and Gag segments) into the chewed-back KpnI/SacI sites ofpBluescriptKS(−). The filled-in MluI/XbaI fragment of pCMV-BL (Moore etal., Anal. Biochem. 247: 203-9 (1997)) was inserted into the chewed-backKpnI/NdeI sites of pNLB-Adapter, replacing lacZ with the CMV promoterand the BL gene (in pNLB, KpnI resides 67 bp upstream of lacZ and NdeIresides 100 bp upstream of the lacZ stop codon), thereby creatingpNLB-Adapter-CMV-BL. To create pNLB-CMV-BL, the HindIII/BlpI insert ofpNLB (containing lacZ) was replaced with the HindIII/BlpI insert ofpNLB-Adapter-CMV-BL. This two step cloning was necessary because directligation of blunt-ended fragments into the HindIII/BlpI sites of pNLByielded mostly rearranged subclones, for unknown reasons.

EXAMPLE 2 Creation of the pNLB-CMV-BL Founder Flock

Sentas and Isoldes were cultured in F10 (Gibco), 5% newborn calf serum(Gibco), 1% chicken serum (Gibco), 50 μg/ml phleomycin (CaylaLaboratories) and 50 μg/ml hygromycin (Sigma). Transduction particleswere produced as described in Cosset et al., 1993, herein incorporatedby reference, with the following exceptions. Two days after transfectionof the retroviral vector pNLB-CMV-BL (from Example 1, above) into 9×10⁵Sentas, virus was harvested in fresh media for 6-16 hours and filtered.All of the media was used to transduce 3×10⁶ Isoldes in 3 100 mm plateswith polybrene added to a final concentration of 4 μg/ml. The followingday the media was replaced with media containing 50 μg/ml phleomycin, 50μg/ml hygromycin and 200 μg/ml G418 (Sigma). After 10-12 days, singleG418 resistant colonies were isolated and transferred to 24-well plates.After 7-10 days, titers from each colony were determined by transductionof Sentas followed by G418 selection. Typically 2 out of 60 coloniesgave titers at 1-3×10⁵. Those colonies were expanded and virusconcentrated to 2-7×10⁶ as described in Allioli et al., Dev. Biol.165:30-7 (1994), herein incorporated by reference. The integrity of theCMV-BL expression cassette was confirmed by assaying for β-lactamase inthe media of cells transduced with NLB-CMV-BL transduction particles.

The transduction vector, pNLB-CMV-BL, was injected into the subgerminalcavity of 546 unincubated SPF White Leghorn embryos, of which 126 chickshatched and were assayed for secretion of β-lactamase (lactamase) intoblood. In order to measure the concentration of active lactamase inunknown samples, a kinetic colorimetric assay was employed in whichPADAC, a purple substrate, is converted to a yellow compoundspecifically by lactamase. Lactamase activity was quantitated bymonitoring the decrease in OD₅₇₀ nm during a standard reaction time andcompared to a standard curve with varying levels of purified lactamase(referred to as the “lactamase assay”). The presence or absence oflactamase in a sample could also be determined by visually scoring forthe conversion of purple to yellow in a test sample overnight or forseveral days (the “overnight lactamase assay”). The latter method wassuitable for detection of very low levels of lactamase or for screeninga large number of samples. At one to four weeks of age, chick serumsamples were tested for the presence of lactamase. Twenty-seven chickshad very low levels of lactamase in their serum that was detectable onlyafter the overnight lactamase assay and, as these birds matured,lactamase was no longer detectable. As shown in Table 1 below and FIG.4A, 9 additional birds (3 males and 6 females) had serum levels oflactamase that ranged from 11.9 to 173.4 ng/ml at six to seven monthspost-hatch.

TABLE 1 Expression of β-Lactamase in NLB-CMV-BL-Transduced ChickensAverage ng/ml of β-Lactamase Egg Egg Serum: 8 Month White: 8 MonthWhite: 14 Sex Band No. Birds Hens³ Month Hens³ NA¹ Controls²  0.0 ± 7.4 0.0 ± 13.6  0.0 ± 8.0 Female 1522 36.7 ± 1.6  56.3 ± 17.8  47.9 ± 14.3Female 1549 11.9 ± 1.3 187.0 ± 32.4 157.0 ± 32.2 Female 1581 31.5 ± 4.8243.8 ± 35.7 321.7 ± 68.8 Female 1587 33.9 ± 1.4 222.6 ± 27.7 291.0 ±27.0 Female 1790 31.0 ± 0.5 136.6 ± 20.2 136.3 ± 11.0 Female 1793 122.8± 3.6  250.0 ± 37.0 232.5 ± 28.6 Male 2395 16.0 ± 2.3 NA NA Male 2421165.5 ± 5.0  NA NA Male 2428 173.4 ± 5.9  NA NA ¹NA: not applicable.²Controls were obtained from untreated hens. ³Represents the average of5 to 20 eggs.

EXAMPLE 3 β-Lactamase Expression in the Egg White of G0 Hens

Fifty-seven pullets transduced with pNLB-CMV-BL retroviral vector wereraised to sexual maturity and egg white from each hen was tested foractive β-lactamase (lactamase) at 8 months of age. Of the 57 birds, sixhad significant levels of lactamase that ranged from 56.3 to 250.0 ng/ml(Table 1, supra). No other hens in this group had detectable levels oflactamase in their egg white, even after incubation of PADAC with thesample for several days. Lactamase was not detectable in egg white from24 hens that were mock injected and in 42 hens that were transduced witha NLB vector that did not carry the lactamase transgene. Stablelactamase expression was still detectable in the egg white of the sixexpressing hens six months following the initial assays (Table 1,supra).

Lactamase was detected in the egg white of all six hens by a westernblot assay with an anti-β-lactamase antibody. The egg white lactamasewas the same size as the bacterially produced, purified lactamase thatwas used as a standard. The amount detected in egg white by Westernanalysis was consistent with that determined by the enzymatic assay,indicating that a significant proportion of the egg white lactamase wasbiologically active. Hen-produced lactamase in egg white stored at 4° C.lost no activity and showed no change in molecular weight even afterseveral months of storage. This observation allowed storage oflactamase-containing eggs for extended periods prior to analysis.

EXAMPLE 4 Germline Transmission and Serum Expression of the D-LactamaseTransgene in G1 and G2 Transgenic Chickens

DNA was extracted from sperm collected from 56 G0 roosters and three ofthe 56 birds that harbored significant levels of the transgene in theirsperm DNA as determined by quantitative PCR were selected for breeding.These roosters were the same three that had the highest levels ofβ-lactamase (lactamase) in their blood (roosters 2395, 2421 and 2428).Rooster 2395 gave rise to three G1 transgenic offspring (out of 422progeny) whereas the other two yielded no transgenic offspring out of630 total progeny. Southern analysis of blood DNA from each of the threeG1 transgenic chickens confirmed that the transgenes were intact andthat they were integrated at unique random loci. The serum of the G1transgenic chicks, 5308, 5657 and 4133, at 6 to 11 weeks post-hatchcontained 0.03, 2.0 and 6.0 μg/ml of lactamase, respectively. The levelsof lactamase dropped to levels of 0.03, 1.1 and 5.0 μg/ml when thechickens were assayed again at 6 to 7 months of age (FIG. 4A).

Hen 5657 and rooster 4133 were bred to non-transgenic chickens to obtainoffspring hemizygous for the transgene. The pedigrees of transgenicchickens bred from rooster 4133 or hen 5657 and the subsequentgenerations are shown in FIG. 5. Transgenic rooster 5308 was also bredbut this bird's progeny exhibited lactamase concentrations that wereeither very low or not detectable in serum and egg white. Activelactamase concentrations in the serum of randomly selected G2 transgenicchicks were measured at 3 to 90 days post-hatch. Of the five G2transgenics bred from hen 5657, all had active lactamase atconcentrations of 1.9 to 2.3 μg/ml (compared to the parental expressionof 1.1 μg/ml, FIG. 4B). All of the samples were collected during thesame period of time, thus, the lactamase concentrations in the serum ofthe offspring were expected to be higher than that of the parent sincethe concentration in hen 5657 had dropped proportionately as shematured. Similarly, the five randomly selected transgenic chicks bredfrom rooster 4133 all had serum lactamase concentrations that weresimilar but higher than that of their parent (FIG. 4B).

EXAMPLE 5 β-Lactamase Expression in the Egg White of Transgenic Hens

Eggs from G1 hen 5657 contained 130 ng of active β-lactamase (lactamase)per ml of egg white (FIG. 6A). Lactamase concentrations were higher inthe first few eggs laid and then reached a plateau that was stable forat least nine months. Eggs from transgenic hens bred from hen 5657 and anon-transgenic rooster had lactamase concentrations that were similar totheir parent (FIG. 6A). Hen 6978 was bred from G2 hen 8617 and siblingG2 rooster 8839 and was homozygous for the transgene as determined byquantitative PCR and Southern analysis. As expected, the concentrationof lactamase in the eggs of bird 6978 was nearly two-fold higher thanher hemizygous parent (FIG. 6B). No other G3 hens bred from hen 5657,were analyzed because hen 6978 was the only female in her clutch. It isimportant to note that the eggs from hens 8867, 8868 and 8869 werecollected eleven months apart and had similar concentrations oflactamase (FIGS. 6A and 6B), again indicating that the expression levelsin the egg white were consistent throughout the lay period.

Rooster 4133 was bred to non-transgenic hens to obtain hemizygous G2hens. Of the 15 transgenic hens analyzed, all had lactamase in the eggwhite at concentrations ranging from 0.47 to 1.34 μg/ml. Fourrepresentative hens are shown in FIG. 7A. When assayed 6 months later,the average expression level had dropped from approximately 1.0 μg/ml to0.8 μg/ml (FIG. 7A). Expression levels were high in the initial eggs andleveled out over several months. After that, the concentrations oflactamase in the eggs remained constant.

G2 hen 8150 and sibling G2 rooster 8191 were crossed to yield hemizygousand homozygous G3 hens. All transgenic G3 hens expressed lactamase inthe white of their eggs at concentrations ranging from 0.52 to 1.65μg/ml (FIG. 7B). The average expression for the G3 hens that werehomozygous was 47% higher than those G2 hens and G3 hens that werehemizygous. The amount of lactamase in the eggs from G2 and G3 hens bredfrom rooster 4133 and his offspring varied significantly (FIGS. 7A and7B), although the levels in the eggs from any given hen in that groupwere relatively constant. The average expression of lactamase wasexpected to double for the homozygous genotype. Western blot analysisconfirmed that the transgene was faithfully producing intact lactamasein the eggs of G2 transgenics. The lactamase level detected on a Westernblot also correlated closely with that determined by the enzyme activityassay, indicating that a significant portion of the egg white lactamasewas bioactive. Thus, retroviral vectors were successfully employed toimplement stable and reliable expression of a transgene in chickens.

Deposition of lactamase in the yolk was detectable but lower than thatof egg white. Seven G2 or G3 hens of rooster 4133's lineage wereanalyzed and the concentration in the yolk ranged from 107 to 375 ng/mlor about 20% the concentration in the egg white. There was nocorrelation between the yolk and egg white lactamase levels of a givenhen (Harvey et al., “Expression of exogenous protein in egg white oftransgenic chickens” (April 2002) Nat. Biotechnol. 20:396-399).

EXAMPLE 6 Production of Founder Males

For pNLB-CMV-BL transduction, freshly laid fertilized White Leghorn eggswere used. Seven to ten microliters of concentrated particles wereinjected into the subgerminal cavity of windowed eggs and chicks hatchedafter sealing the window. 546 eggs were injected. Blood DNA wasextracted and analyzed for the presence of the transgene using aprobe-primer set designed to detect the neo resistance gene via theTaqman assay. As can be seen in Table 2 below, approximately 25% of allchicks had detectable levels of transgene in their blood DNA.

TABLE 2 Summary of Transgenesis with the NLB-CMV-BL Vectors TransgeneNLB-CMV-BL Production of Number of injections 546 G0 founder Number ofbirds hatched (%) 126 (23.1%)  flock Number of chicks with transgene in36 (28.6%) their blood DNA (%) Number of males 56 Number of males withtransgene in 3 (5.4%) their sperm DNA (%) Number of males thattransmitted 1 (1.8%) transgene to progeny (%) Production of Number ofchicks bred from G0 males 1026 G1 flock Number of G1 transgenics 3 Rateof germline transmission 0.29% Production of Number of chicks bred fromG1 120 G2 flock transgenics Number of G2 transgenics 61 Rate of germlinetransmission 50.8%

EXAMPLE 7 Germline Transmission of the Transgene

Taqman detection of the neo resistance gene in sperm DNA was used toidentify candidate G0 males for breeding. Three G0 males wereidentified, wherein each had the NLB-CMV-BL transgene in their sperm DNAat levels that were above background. All G0 males positive for thetransgene in their sperm were bred to non-transgenic hens to identifyfully transgenic G1 offspring.

For NLB-CMV-BL 1026 chicks were bred, respectively, and three G1 chicksobtained for each transgene (Table 2, supra). All G1 progeny came fromthe male with the highest level of transgene in his sperm DNA, eventhough an equivalent number of chicks were bred from each male.

EXAMPLE 8 Southern Analysis of G1s and G2s

In order to confirm integration and integrity of the inserted vectorsequences, Southern blot analysis was performed on DNA from G1 and G2transgenics. Blood DNA was digested with HindIII and hybridized to a neoresistance probe to detect junction fragments created by the internalHindIII site found in the pNLB-CMV-BL vector (FIG. 3B) and genomic sitesflanking the site of integration. Each of the 3 G1 birds carryingNLB-CMV-BL had a junction fragment of unique size, indicating that thetransgene had integrated into three different genomic sites. G1s werebred to non-transgenic hens to obtain hemizygous G2s. As can be seen inTable 2 (supra), 50.8% of offspring from G1 roosters harboringNLB-CMV-BL were transgenic as expected for Mendelian segregation of asingle integrated transgene. Southern analysis of HindIII-digested DNAfrom G2 offspring detected junction fragments similar in size to thoseoriginating from their transgenic parents, indicating that the transgenewas transmitted intact.

EXAMPLE 9 Screening for G3 Progeny Homozygous for the Transgene

In order to obtain transgenic chickens homozygous for the transgene, G2hemizygous birds having NLB-CMV-BL integrated at the same site (e.g.,progeny of the same G1 male) were crossbred. Two groups were bred: thefirst was a hen and rooster arising from the G1 4133 male and the secondfrom the G1 5657 hen. The Taqman assay was used to quantitatively detectthe neo resistance transgene in G3 progeny using a standard curve. Thestandard curve was constructed using known amounts of genomic DNA fromthe G1 transgenic 4133 male hemizygous for the transgene as determinedby Southern analysis. The standard curve ranged from 1 to 1.6×10⁴ totalcopies of the transgene or 0.2 to 3.1 transgene copies per diploidgenome. Because reaction components were not limited during theexponential phase, amplification was very efficient and gavereproducible values for a given copy number. There was a reproducible,one-cycle difference between each standard curve differing two-fold incopy number.

In order to determine the number of transgene alleles in the G3offspring, DNAs were amplified and compared to the standards. DNA fromnon-transgenics did not amplify. Birds homozygous for the transgenicallele gave rise to plots initiating the amplification one cycle earlierthan those hemizygous for the allele. The sequence detection program wasable to calculate the number of alleles in an unknown DNA sample basedon the standard curve and the cycle threshold (Ct) at which a sample'samplification plot exhibited a significant rise. The data are shown inTable 3 below.

In order to confirm Taqman copy number analysis, DNA of selected birdswas analyzed by Southern blotting using PstI-digested DNA and a probecomplementary to the neo resistance gene to detect a 0.9 kb fragment.Detection of a small fragment was chosen since transfer of smaller DNAsfrom gel to membrane is more quantitative. The signal intensity of the0.9 kb band corresponded well to the copy number of G3 transgenic birdsas determined by the Taqman assay. The copy numbers of an additionaleighteen G3 transgenic birds analyzed by Southern blotting were alsoconsistent with that determined by Taqman. A total of 33 progeny wereanalyzed for the 4133 lineage, of which 9 (27.3%) were non-transgenic,16 (48.5%) were hemizygous and 8 (24.2%) were homozygous. A total of 10progeny were analyzed for the 5657 lineage, of which 5 (50.0%) werenon-transgenic, 1 (10.0%) was hemizygous and 4 (40.0%) were homozygous.The observed ratio of non-transgenics, hemizygotes and homozygotes forthe 4133 lineage G3 progeny was not statistically different from theexpected 1:2:1 ratio as determined by the χ2 test (P is less than orequal to 0.05). Progeny of the 5657 lineage did not have the expecteddistribution but this could have been due to the low number of progenytested (Harvey et al., “Consistent production of transgenic chickensusing replication deficient retroviral vectors and high-throughputscreening procedures” (February 2002) Poultry Science 81:202-212).

TABLE 3 Determination of Transgene Copy Number in G3 Offspring Bred fromG2 Transgenics Band No. Copies per (Std. No. or Mean Total StandardDiploid G1 Parent NTC¹) Ct² Copy Number Deviation Genome³ NA⁴ 4133 27.33,975 145.7 1 4133 6792 40.0 0 0.0 0 5657 6977 25.9 10,510 587.0 2 56576978 25.8 10,401 505.1 2 4133 7020 26.7 6,064 443.1 1 4133 7021 26.85,239 133.8 1 4133 7022 26.1 9,096 352.3 2 4133 7023 26.8 5,424 55.7 14133 7024 26.9 4,820 110.1 1 5657 7110 26.4 8,092 1037.5 2 5657 711130.4 403 46.3 0 5657 7112 33.2 60 6.1 0 4133 7142 26.5 6,023 367.6 14133 7143 25.9 9,474 569.8 2 4133 7144 25.7 12,420 807.7 2 4133 733827.2 4,246 201.7 1 5657 7407 37.7 1 1.0 0 NA (std1) 29.1 1,000 0.0 0.2NA (std2) 28.1 2,000 0.0 0.4 NA (std3) 27.1 4,000 0.0 0.8 NA (std4) 26.28,000 0.0 1.6 NA (std5) 25.3 16,000 0.0 3.1 NA (NTC) 39.8 −1 0.0 0.0¹Std. No.: standard number; NTC: no template control. ²Ct: cyclethreshold; cycle at which a sample's fluorescence exhibited asignificant increase above background. ³Copies per diploid genome weredetermined by dividing the mean by 5100 and rounding to the nearestfirst decimal place. ⁴NA: not applicable.

EXAMPLE 10 Vector Construction for pNLB-MDOT-EPO Vector

Following the teachings of Example 1 (Vector Construction) of thespecification, an pNLB-MDOT-EPO vector was created, substituting an EPOencoding sequence for the BL encoding sequence (FIG. 8B). Instead ofusing the CMV promoter, MDOT was used (FIG. 13). MDOT is a syntheticpromoter which contains elements from both the ovomucoid (MD) andovotransferrin (TO) promoter. (pNLB-MDOT-EPO vector, a.k.a.pAVIJCR-A145.27.2.2).

The DNA sequence for human EPO based on hen oviduct optimized codonusage was created using the BACKTRANSLATE program of the WisconsinPackage, version 9.1 (Genetics Computer Group, Inc., Madison, Wis.) witha codon usage table compiled from the chicken (Gallus gallus) ovalbumin,lysozyme, ovomucoid, and ovotransferrin proteins. The DNA sequence wassynthesized and cloned into the 3′ overhang T's of pCRII-TOPO(Invitrogen) by Integrated DNA Technologies, Coralville, Iowa, on acontractual basis. The EPO coding sequence was then removed from pEpoMMwith Hind III and Fse I, purified from a 0.8% agarose-TAE Gel, andligated to Hind III and Fse I digested, alkaline phosphatase-treatedpCMV-IFNMM. The resulting plasmid was pAVIJCR-A137.43.2.2 whichcontained the EPO coding sequence controlled by the cytomegalovirusimmediate early promoter/enhancer and SV40 polyA site. The plasmidpAVIJCR-A137.43.2.2 was digested with Nco I and Fse I and theappropriate fragment ligated to an Nco I and Fse I-digested fragment ofpMDOTIFN to obtain pAVIJCR-A137.87.2.1 which contained EPO driven by theMDOT promoter. In order to clone the EPO coding sequence controlled bythe MDOT promoter into the NLB retroviral plasmid, the plasmidspALVMDOTIFN and pAVIJCR-A137.87.2.1 were digested with Kpn I and Fse I.Appropriate DNA fragments were purified on a 0.8% agarose-TAE gel, thenligated and transformed into DH5α cells. The resulting plasmid waspNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2).

EXAMPLE 11 Production of Transgenic Chickens and Fully Transgenic G1Chickens Expressing EPO

Production of NLB-MDOT-EPO transduction particles were performed asdescribed for NLB-CMV-BL (see Example 2). Approximately 300 WhiteLeghorn eggs were windowed according to the Speksnijder procedure (U.S.Pat. No. 5,897,998), then injected with about 7×10⁴ transducingparticles per egg. Eggs hatched 21 days after injection, and human EPOlevels were measured by EPO ELISA from serum samples collected fromchicks one week after hatch.

In order to screen for G0 roosters which contained the EPO transgene intheir sperm, DNA was extracted from rooster sperm samples by Chelex-100extraction (Walsh et al., 1991). DNA samples were then subjected toTaqman® analysis on a 7700 Sequence Detector (Perkin Elmer) using the“neo for-1” (5′-TGGATTGCACGCAGGTTCT-3′; SEQ ID NO: 5) and “neo rev-1”(5′-TGCCCAGTCATAGCCGAAT-3′; SEQ ID NO: 6) primers and FAM labeledNEO-PROBE1 (5′-CCTCTCCACCCAAGCGGCCG-3′; SEQ ID NO: 7) to detect thetransgene. Eight G0 roosters with the highest levels of the transgene intheir sperm samples were bred to nontransgenic SPAFAS (White Leghorn)hens by artificial insemination. Blood DNA samples were screened for thepresence of the transgene by Taqman® analysis as described above.

Out of 1,054 offspring, 16 chicks were found to be transgenic (G1avians). Chick serum was tested for the presence of human EPO by EPOELISA, and EPO was present at about 70 nanogram/ml (ng/ml). Egg white ineggs from G1 hens was also tested for the presence of human EPO by EPOELISA and found to contain human EPO at about 70 ng/ml. The EPO presentin eggs (i.e., derived from the optimized coding sequence of human EPO)was found to be biologically active when tested on a human EPOresponsive cell line (HCD57 murine erythroid cells) in a cell cultureassay.

EXAMPLE 12 Vector Construction for pNLB-CMV-IFN

Following the teachings of Example 1, a pNLB-CMV-IFN vector was created(FIG. 8A), substituting an IFN encoding sequence for the BL encodingsequence of Example 1.

An optimized coding sequence was created, wherein the most frequentlyused codons for each particular amino acid found in the egg whiteproteins ovalbumin, lysozyme, ovomucoid, and ovotransferrin were used inthe design of the optimized human IFN-α 2b coding sequence that wasinserted into vectors of the present invention. More specifically, theDNA sequence for optimized human IFN-α 2b is based on the hen oviductoptimized codon usage and was created using the BACKTRANSLATE program ofthe Wisconsin Package, Version 9.1 (Genetics Computer Group Inc.,Madison, Wis.) with a codon usage table compiled from the chicken(Gallus gallus) ovalbumin, lysozyme, ovomucoid, and ovotransferrinproteins. For example, the percent usage for the four codons of theamino acid alanine in the four egg white proteins is 34% for GCU, 31%for GCC, 26% for GCA, and 8% for GCG. Therefore, GCU was used as thecodon for the majority of alanines in the optimized human IFN-α 2bcoding sequence. The vectors containing the gene for optimized humanIFN-α 2b were used to create transgenic avians that express transgenicpoultry derived interferon-α 2b (TPD IFN-α 2b) in their tissues andeggs.

The template and primer oligonucleotides listed in Table 4 below wereamplified by PCR with Pfu polymerase (Stratagene, La Jolla, Calif.)using 20 cycles of 94° C. for 1 min.; 50° C. for 30 sec.; and 72° C. for1 min. and 10 sec. PCR products were purified from a 12%polyacrylamide-TBE gel by the “crush and soak” method (Maniatis et al.1982), then combined as templates in an amplification reaction usingonly IFN-1 and IFN-8 as primers (see Table 4). The resulting PCR productwas digested with Hind III and Xba I and gel purified from a 2%agarose-TAE gel, then ligated into Hind III and Xba I digested, alkalinephosphatase-treated pBluescript KS (Stratagene), resulting in theplasmid pBluKSP-IFNMagMax. Both strands were sequenced by cyclesequencing on an ABI PRISM 377 DNA Sequencer (Perkin-Elmer, Foster City,Calif.) using universal T7 or T3 primers. Mutations in pBluKSP-IFNderived from the original oligonucleotide templates were corrected bysite-directed mutagenesis with the Transformer Site-Directed MutagenesisKit (Clontech, Palo Alto, Calif.). The IFN coding sequence was thenremoved from the corrected pBluKSP-IFN with Hind III and Xba 1, purifiedfrom a 0.8% agarose-TAE Gel, and ligated to Hind III and Xba I digested,alkaline phosphatase-treated pCMV-BetaLa-3B-dH. The resulting plasmidwas pCMV-IFN which contained an IFN coding sequence controlled by thecytomegalovirus immediate early promoter/enhancer and SV40 polyA site.In order to clone the IFN coding sequence controlled by the CMVpromoter/enhancer into the NLB retroviral plasmid, pCMV-IFN was firstdigested with ClaI and XbaI, then both ends were filled in with Klenowfragment of DNA polymerase (New England BioLabs, Beverly, Mass.).pNLB-adapter was digested with NdeI and KpnI, and both ends were madeblunt by T4 DNA polymerase (New England BioLabs). Appropriate DNAfragments were purified on a 0.8% agarose-TAE gel, then ligated andtransformed into DH5α cells. The resulting plasmid waspNLB-adapter-CMV-IFN. This plasmid was then digested with MluI andpartially digested with BlpI and the appropriate fragment was gelpurified. pNLB-CMV-EGFP was digested with MluI and BlpI, thenalkaline-phosphatase treated and gel purified. The MluI/BlpI partialfragment of pNLB-adapter-CMV-IFN was ligated to the large fragmentderived from the MluI/BlpI digest of pNLB-CMV-EGFP creatingpNLB-CMV-IFN.

TABLE 4 Sequence Primer Primer of Primer Template Sequence of Template 1Sequence of Primer 1 2 2 IFN-A 5′ATGGCTTTGACCTTTGCCTTACTG IFN-15′CCCAAGCTTTCACCATGG IFN-2 5′CTGTG SEQ ID GTGGCTCTCCTGGTGCTGAGCTGCA SEQID CTTTGACCTTTGCCTT3′ SEQ ID GGTCTGA NO: 8 AGAGCAGCTGCTCTGTGGGCTGCG NO:9 NO: 10 GGCAGA ATCTGCCTCA3′ T3′ IFN-B 5′GACCCACAGCCTGGGCAGCAGGA IFN-2b5′ATCTGCCTCAGACCCACA IFN-3b 5′AACTC SEQ ID GGACCCTGATGCTGCTGGCTCAGAT SEQID G3′ SEQ ID CTCTTGA NO: 11 GAGGAGAATCAGCCTGTTTAGCTG NO: 12 NO: 13GGAAAG CCTGAAGGATAGGCACGATTTTGG CCAAAAT CTTT3′ C3′ IFN-C5′CTCAAGAGGAGTTTGGCAACCAG IFN-3c 5′GATTTTGGCTTTCCTCAA IFN-4 5′ATCTGC SEQID TTTCAGAAGGCTGAGACCATCCCTG SEQ ID GAGGAGTT3′ SEQ ID TGGATCA NO: 14TGCTGCACGAGATG3′ NO: 15 NO: 16 TCTCGTG C3′ IFN-D5′ATCCAGCAGATCTTTAACCTGTTT IFN-4b 5′GCACGAGATGATCCAGC IFN-5 5′ATCGTT SEQID AGCACCAAGGATAGCAGCGCTGCT SEQ ID AGAT3′ SEQ ID CAGCTGC NO: 14TGGGATGAGACCCTGCTGGATAAG NO: 18 NO: 19 TGGTACA TTTTACACCGAGCTGTACCAGCA3′3′ IFN-E 5′GCTGAACGATCTGGAGGCTTGCG IFN-5b 5′TGTACCAGCAGCTGAAC IFN-65′CCTCAC SEQ ID TGATCCAGGGCGTGGGCGTGACCG SEQ ID GAT 3′ SEQ ID AGCCAG NO:20 AGACCCCTCTGATGAAGGAGGATA NO: 21 NO: 22 GATGCTA GCATCCT3′ T3′ IFN-F5′GGCTGTGAGGAAGTACTTTCAGA IFN-6b 5′ATAGCATCCTGGCTGTGA IFN-7 5′ATGAT SEQID GGATCACCCTGTACCTGAAGGAGA SEQ ID GG 3′ SEQ ID CTCAGCC NO: 23AGAAGTACAGCCCTTGCGCTTGGG NO: 24 NO: 25 CTCACGA AAGTCGTGAGGG3′ C3′ IFN-G5′CTGAGATCATGAGGAGCTTTAGC IFN-7b 5′GTCGTGAGGGCTGAGAT IFN-8 5′TGCTCT SEQID CTGAGCACCAACCTGCAAGAGAGC SEQ ID CAT 3′ SEQ ID AGACTTT NO: 26TTGAGGTCTAAGGAGTAA3′ NO: 27 NO: 28 TTACTCC TTAGACC TCAAGCT CT3′

EXAMPLE 13 Production of Transgenic Chickens and Fully Transgenic G1Chickens Expressing IFN

Transduction particles of pNLB-CMV-IFN were produced following theprocedures of Example 2. Approximately 300 White Leghorn (strain Line 0)eggs were windowed according to the Speksnijder procedure (U.S. Pat. No.5,897,998), then injected with about 7×10⁴ transducing particles peregg. Eggs hatched 21 days after injection, and human IFN levels weremeasured by IFN ELISA from serum samples collected from chicks one weekafter hatch.

In order to screen for G0 roosters which contained the IFN transgene intheir sperm, DNA was extracted from rooster sperm samples by Chelex-100extraction (Walsh et al., 1991). DNA samples were then subjected toTaqman® analysis on a 7700 Sequence Detector (Perkin Elmer) using the“neo for-1” (5′-TGGATTGCACGCAGGTTCT-3′; SEQ ID NO: 5) and “neo rev-1”(5′-GTGCCCAGTCATAGCCGAAT-3′; SEQ ID NO: 6) primers and FAM labeledNEO-PROBE1 (5′-CCTCTCCACCCAAGCGGCCG-3′; SEQ ID NO: 7) to detect thetransgene. Three G0 roosters with the highest levels of the transgene intheir sperm samples were bred to nontransgenic SPAFAS (White Leghorn)hens by artificial insemination.

Blood DNA samples were screened for the presence of the transgene byTaqman® analysis as described above. Out of 1,597 offspring, one roosterwas found to be transgenic (a.k.a. “Alphie”). Alphie's serum was testedfor the presence of hIFN by hIFN ELISA, and hIFN was present at 200ng/ml.

Alphie's sperm was used for artificial insemination of nontransgenicSPAFAS (White Leghorn) hens. 106 out of 202 (about 52%) offspringcontained the transgene as detected by Taqman® analysis. These breedingresults followed a Mendelian inheritance pattern and indicated thatAlphie is transgenic.

EXAMPLE 14 Carbohydrate Analysis of Transgenic Poultry DerivedInterferon-α 2b (TPD IFN-α 2b)

Experimental evidence revealed a new glycosylation pattern ininterferon-α 2b derived from avians (i.e., TPD IFN-α 2b). TPD IFN-α 2bwas found to contain two new glyco forms (bands 4 and 5 are α-Galextended disaccharides; see FIG. 9) not normally seen in humanperipheral blood leukocyte derived interferon-α 2b (PBL IFN-α 2b) ornatural human interferon-α 2b (natural hIFN). TPD IFN-α 2b was alsofound to contain O-linked carbohydrate structures that are similar tohuman PBL IFN-α 2b and was more efficiently produced in chickens thenthe human form.

The coding sequence for human IFN-α 2b was optimized (Example 12, supra)resulting in a recombinant IFN-α 2b coding sequence. TPD IFN-α 2b wasthen produced in chickens (Example 13, supra). A carbohydrate analysis,including a monosaccharide analysis and FACE analysis, revealed thesugar make-up or novel glycosylation pattern of the protein. As such,TPD IFN-α 2b showed the following monosaccharide residues:N-Acetyl-Galactosamine (NAcGal), Galactose (Gal), N-Acetyl-Glucosamine(NAcGlu), and Sialic acid (SA). No N-linked glycosylation was found inTPD IFN-α 2b. Instead, TPD IFN-α 2b was found to be O-glycosylated atThr-106. This type of glycosylation is similar to human IFN-α 2, whereinthe Thr residue at position 106 is unique to IFN-α 2. In addition, TPDIFN-α 2b was found to have no mannose residues. A FACE analysis revealed6 bands (FIG. 9) that represent various sugar residues, wherein bands 1,2 and 3 are un-sialylated, mono-sialylated, and di-sialylated,respectively (FIG. 10). The sialic acid (SA) linkage is alpha 2-3 toGalactose (Gal) and alpha 2-6 to N-Acetyl-Galactosamine (NAcGal). Band 6represents an un-sialylated tetrasaccharide. Bands 4 and 5 were found tobe alpha-Galactose (alpha-Gal) extended disaccharides that are not seenin human PBL IFN-α 2b. FIG. 10 shows the comparison of TPD IFN-α 2b (eggwhite HIFN) and human PBL IFN-α 2b (natural HIFN). Minor bands werepresent between bands 3 and 4 and between bands 4 and 5 in TPD IFN-α 2b(vide infra).

The protein was found to be O-glycosylated at Thr-106 with specificresidues, such as:

wherein

-   Gal=Galactose,-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.

The percentages were as follows:

Minor bands were present between bands 3 and 4 and between bands 4 and 5which account for about 17% in TPD IFN-α 2b.

EXAMPLE 15 Expression of MAbs from Plasmid Transfection and RetroviralTransduction Using the EMCV IRES in Avian Cells

The light chain (LC) and heavy chain (HC) of a human monoclonal antibodywere expressed from a single vector, pCMV-LC-emcvIRES-HC, by placementof an IRES from the encephalomyocarditis virus (EMCV) (see also Jang etal. (1988) “A segment of the 5′ nontranslated region ofencephalomyocarditis virus RNA directs internal entry of ribosomesduring in vitro translation” J. Virol. 62:2636-2643) between the LC andHC coding sequences. Transcription was driven by the CMV promoter.

In order to test expression of monoclonal antibodies from two separatevectors, the LC or HC linked to the CMV promoter were cotransfected intoLMH/2a cells, an estrogen-responsive, chicken hepatocyte cell line (seealso Binder et al. (1990) “Expression of endogenous and transfectedapolipoprotein II and vitellogenin II genes in an estrogen responsivechicken liver cell line” Mol. Endocrinol. 4:201-208). Contransfection ofpCMV-LC and pCMV-HC resulted in 392 ng/ml of MAbs determined by a MAbELISA whereas transfection of pCMV-LC-emcvIRES-HC resulted in 185 ng/mlof MAb.

The CMV-LC-emcv-HC cassette was inserted in a retroviral vector based onthe Moloney murine leukemia virus (MLV), creatingpL-CMV-LC-emcvIRES-HC-RN-BG. LMH cells (see also Kawaguchi et al. (1987)“Establishment and characterization of a chicken hepatocellularcarcinoma cell line, LMH” Cancer Res. 47:4460-4464), the parent line ofLMH/2a, were used as target cells because they are not neomycinresistant. LMH cells were transduced with the L-CMV-LC-emcvIRES-HC-RN-BGretroviral vector and selected with neomycin and passaged for severalweeks. LMH cells were separately transduced and neomycin selected withthe parent MLV vector, LXRN. Media from LXRN cells were negative forMAb, whereas media from the L-CMV-LC-emcvIRES-HC-RN-BG-transduced cellscontained 22 ng/ml of MAb.

EXAMPLE 16 Production of Transgenic Chickens and Fully Transgenic G1Chickens Expressing MAbs

A pNLB-CMV-LC-emcv-HC vector is produced by substituting theCMV-LC-emcv-HC cassette of Example 15 for the CMV-BL cassette ofpNLB-CMV-BL of Example 1.

Transduction particles of pNLB-CMV-LC-emcv-HC are produced following theprocedures of Example 2. Approximately 300 White Leghorn (strain Line 0)eggs are windowed according to the Speksnijder procedure (U.S. Pat. No.5,897,998) and are then injected with about 7×10⁴ transducing particlesper egg. Eggs hatch 21 days after injection, and human MAb levels aremeasured by ELISA from serum samples collected from chicks one weekafter hatch.

G0 roster which contain the transgene in their sperm are identified byTaqman® analysis. Three G0 roosters with the highest levels of thetransgene in their sperm samples are bred to nontransgenic SPAFAS (WhiteLeghorn) hens by artificial insemination.

Over 1000 offspring are screened and more than 10 chicks are found to betransgenic (G1 avians). Chick serum is tested for the presence of theMAb by ELISA. The MAb is found to be present in an amount greater than10 μg/ml of serum. Egg white in eggs from G1 hens is also tested for thepresence of the MAb by ELISA and is found to be present in an amountgreater than 10 μg/ml of egg white.

EXAMPLE 17 Construction of pNLB-CMV-hG-CSF

This vector construction effectively replaces the IFN coding region ofthe pNLB-CMV-IFN vector of Example 12 with the coding sequence of G-CSF.The hG-CSF ORF (human granulocyte colony stimulating factor open readingframe) was amplified from pORF9-hG-CSFb (cat. no. porf-hgcsfb,Invivogen, San Diego, Calif.) with the primers 5′GCSF(ggggggaagctttcaccatggctggacctgcca; SEQ ID NO: 32) and 3′GCSF(actagacttttcagggctgggcaaggtggcg; SEQ ID NO: 33) to create a 642 basepair (bp) PCR product. In order to provide the pNLB-CMV-hG-CSF constructwith a sequence 3′ of the G-CSF coding sequence identical to that foundin pNLB-CMV-IFN alpha-2b, an 86 bp fragment of pNLB-CMV-IFN alpha-2b,which is present adjacent to the 3′ end of the INF coding sequence, wasamplified by PCR using the primers 5′GCSF-NLB(ccagccctgaaaagtctagtatggggattggtg; SEQ ID NO: 34) and 3′GCSF-NLB(gggggggctcagctggaattccgcc; SEQ ID NO: 35). The two PCR products (642 bpand 86 bp) were mixed and fused by PCR amplification with primers 5′GCSFand 3′GCSF-NLB. The PCR product was cloned into pCR®4Blunt-TOPO® plasmidvector (Invitrogen) according to the manufacturer's instructions andelectroporated into DH5α-E cells, producing pFusion-hG-CSF-NLB.pFusion-hG-CSF-NLB was digested with Hind III and Blp I and the 690 bpG-CSF fragment was gel purified. The IFN alpha-2b coding sequence wasremoved from pNLB-CMV-IFN alpha-2b by digesting with Blp I. The vectorwas then religated and clones were selected which lacked the IFN codinginsert, creating pNLB-CMV-delta hIFN alpha-2b. pNLB-CMV-delta IFNalpha-2b was digested with Blp I and partially digested with Hind IIIand the 8732 bp Blp I-Hind III vector fragment was gel purified. The8732 bp fragment was ligated to the 690 bp Hind III/Blp I G-CSF fragmentto create pNLB-CMV-G-CSF. The G-CSF ORF was verified by sequencing.

EXAMPLE 18 Production of Transgenic Chickens Expressing HumanGranulocyte Colony Stimulating Factor (hG-CSF)

Production of NLB-CMV-hG-CSF transduction particles was performed asdescribed for NLB-CMV-BL in Example 2. The embryos of 277 stage X eggswere injected with 7 μl of NLB-CMV-hG-CSF transduction particles (titerswere 2.1×10⁷-6.9×10⁷). 86 chicks hatched and were raised to sexualmaturity. 60 chicks tested positive for G-CSF which were evenly dividedin sex; 30 male and 30 females. Egg white from 21 hens was assayed byELISA for the presence of hG-CSF. Five hens were found to havesignificant levels of hG-CSF protein in the egg white at levels thatranged from 0.05 ug/ml to 0.5 μg/ml.

DNA was extracted from rooster sperm samples by Chelex-100 extraction(Walsh et al., 1991). DNA samples were then subjected to Taqman™analysis on a 7700 Sequence Detector (Perkin Elmer) using the primersSJ-G-CSF for (cagagcttcctgctcaagtgctta; SEQ ID NO: 36) and SJ-G-CSF rev(ttgtaggtggcacacagcttct; SEQ ID NO: 37) and the probe, SJ-G-CSF probe(agcaagtgaggaagatccagggcg; SEQ ID NO: 38), to detect the transgene. Therooster with the highest levels of the transgene in his sperm sampleswas bred to nontransgenic SPAFAS (White Leghorn) hens by artificialinsemination.

Blood DNA samples were screened for the presence of the transgene byTaqman™ analysis as described above. Out of 2264 offspring, 13 G1s werefound to be transgenic and each were serum positive for the presence ofG-CSF with one hen (XGF498) having approximately 136.5 ng/ml G-CSF inthe serum and 5.6 μg/ml G-CSF in the egg white, each as measured byELISA.

Two G1 roosters (QGF910 and DD9027) which were of the same line asXGF498 (therefore having the identical transgene inserted into identicalposition in the genome) were crossed with nontransgenic hens, to producefemale offspring that lay eggs containing poultry derived G-CSF.Milligram quantities of the G-CSF were purified from egg white collectedfrom eggs of QGF910 and DD9027 offspring. Patterns of representativeglycosylation structures of the poultry derived G-CSF were determinedfrom the G-CSF obtained as disclosed in Example 20.

EXAMPLE 19 Production of Transgenic Chickens Expressing Human CytotoxicLymphocyte Antigen Four-Fc Fusion Protein (CTLA4-Fc)

pNLB-1.8OM-CTLA4Fc and pNLB-3.9OM-CTLA4Fc were constructed as disclosedin U.S. patent application Ser. No. 11/047,184, filed Jan. 31, 2005, thedisclosure of which is incorporated in its entirety herein by reference.Production of pNLB-1.8OM-CTLA4Fc and pNLB-3.9OM-CTLA4Fc transductionparticles were performed as described for pNLB-CMV-BL in Example 2. 193white leghorn eggs were injected with 7 μl of pNLB-1.8OM-CTLA4Fctransduction particles (titers were ˜4×10⁶) and 72 chicks hatched. 199white leghorn eggs were injected with 7 μl of pNLB-3.9OM-CTLA4Fctransduction particles (titers were ˜4×10⁶) and 20 chicks hatched.

Egg white from 30 hens produced with the pNLB-1.8OM-CTLA4Fc particleswere assayed by ELISA for the presence of CTLA4-Fc. One hen was found tohave significant levels of CTLA4-Fc protein in the egg white at anaverage level of 0.132 μg/ml (5 eggs assayed).

Egg white from seven hens produced with the pNLB-3.9OM-CTLA4Fc particleswere assayed by ELISA for the presence of CTLA4-Fc. Two hens were foundto have significant levels of CTLA4-Fc protein in the egg white at anaverage level of 0.164 μg/ml (5 eggs assayed) for one hen and an averagelevel of 0.123 μg/ml (5 eggs assayed) for the second positive hen.

EXAMPLE 20 Carbohydrate Analysis of Transgenic Poultry Derived G-CSF

The TPD G-CSF oligosaccharide structures were determined by employingthe following analysis techniques as are well known to practitioners ofskill in the art. MALDI-TOF-MS (Matrix assisted laser desorptionionization time-of-flight mass spectrometry) analysis and ESI MS/MS(electrospray ionization tandem mass spectrometry) were performed on theoligosaccharides after release from the peptide backbone. The O-linkedoligosaccharides were chemically released from the protein and werepermethylated using the NaOH method involving reaction with methyliodide under anhydrous DMSO and extracted into chloroform prior toanalysis. Direct mass spectrometry was performed on the intactglycosylated G-CSF. Analyses were also performed on the polysaccharidestructures using HPLC analysis. Briefly, after release from the proteinbackbone the structures were separated using HPLC. Samples of theindividual polysaccharide species were digested with certain enzymes andthe digest products were analyzed by HPLC providing for structuredetermination as is understood in the art.

The structures as determined are shown below. Interestingly, Structure Cand Structure D may be precursor forms of Structure E shown below. Ithas been estimated, the invention not being limited thereto, thatstructure A is present on the poultry derived glycosylated G-CSF about20% to about 40% of the time and that structure B is present on thepoultry derived glycosylated G-CSF about 5% to about 25% of the time andthat structure C is present on the poultry derived glycosylated G-CSFabout 10% to about 20% of the time and that structure D is present onthe poultry derived glycosylated G-CSF about 5% to about 15% of the timeand that structure E is present on the poultry derived glycosylatedG-CSF about 1% to about 5% of the time and that structure F is presenton the poultry derived glycosylated G-CSF about 10% to about 25% of thetime and that structure G is present on the poultry derived glycosylatedG-CSF about 20% to about 30% of the time.

Monosaccharide analysis was performed by GC/MS (gas chromatography-massspectrometry) on poultry derived G-CSF that had been spiked withArabitol (internal standard), hydrolyzed, N-acetylated and TMSderivatized using methods readily available to those skilled in the art.The derivatized sample was compared to a standard mixture of sugarssimilarly derivatized. Sialic acid analysis of the poultry derived G-CSFwas performed after spiking with ketodeoxynonulosonic acid, lyophilizedthen hydrolyzed, desalted and re-lyophilized. Analysis of the sample wasperformed on a Dionex BioLC system using appropriate standards. Theseanalyses showed the presence of galactose, glucose,N-acetylgalactosamine, N-acetylglucosamine and sialic acid(N-acetylneuraminic acid) as seen in Table 5. The data in Table 5supersedes preliminary data generated by HPAEC-PAD analysis whichdetermined a greater percentage of N-acetylglucosamine to be present.

TABLE 5 TPD G-CSF Nmoles Nmoles Monosaccharide detected Detected/mgGalactose 4.5 34.5 N-Acetylgalactosamine 2.9 22.2 N-Acetylglucosamine0.95 7.3 Sialic Acid 6.0 46.0

Linkage analysis was performed on a permethylated glycan sample of thepoultry derived G-CSF that was hydrolyzed in TFA and reduced in sodiumborodeuteride. The borate was removed by three additions ofmethanol:glacial acetic acid (9:1) followed by lyophilization and thenacetylation by acetic anhydride. After purification by extraction withchloroform, the sample was examined by GC/MS. A mixture of standards wasalso run under the same conditions. The linkages were determined asfollows:

-   i. The sialic acid linkage is 2-3 to galactose and 2-6 to    N-acetylgalactosamine-   ii. The galactose linkage is 2-3 to N-acetylgalactosamine and 2-4 to    N-acetylglucosamine-   iii. The N-acetylglucosamine linkage is 2-6 to N-acetylgalactosamine

EXAMPLE 21 In Vitro Cell Proliferation Activity of Poultry Derived G-CSF(TPD G-CSF)

The in vitro biological activity of TPD G-CSF was demonstrated using theNFS-60 cell proliferation assay. Briefly, NFS-60 cells were maintainedin growth media containing GM-CSF. Confluent cultures were harvested,washed and plated at a cell density of 10⁵ cells per well with growthmedia alone. TPD G-CSF and bacterial derived human G-CSF (i.e.,Neupogen®) were serial diluted in growth media and added to separatewells in triplicate. Cell proliferation was determined by metabolicreduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and was quantified spectrophotometrically. The specificactivity of the avian derived G-CSF was determined by comparing the ED₅₀of Neupogen® with that of the purified avian derived G-CSF. The specificactivity of TPD G-CSF over a 14 day period was determined to be well inexcess of that of the bacterial derived G-CSF Neupogen®(non-glycosylated G-CSF). See FIG. 17.

EXAMPLE 22 Construction of pNLB-CMV-Des-Arg166-EPO

pNLB-CMV-IFN described in Example 12 was digested with Hind III andEcoRI in order to replace the hIFN α2 coding sequence and signal peptidecoding sequence with an EPO coding sequence plus signal peptide (SEQ IDNO: 42) shown below. Because multiple EcoRI and Hind III sites exist inthe vector, RecA-assisted restriction endonuclease (RARE) cleavagemethod was used to cut the desired sites. The following oligonucleotideswere used in the RARE procedure:

-   pnlbEcoRI3805rare (5′-GAC TCC TGG AGC CCG TCA GTA TCG GCG GAA TTC    CAG CTG AGC GCC GGT CGC TAC CAT TAC-3′) (SEQ ID NO: 43) and-   pnlbHinD III3172rare (5′-TAA TAC GAC TCA CTA TAG GGA GAC CGG AAG CTT    TCA CCA TGG CTT TGA CCT TTG CCT TAC-3′) (SEQ ID NO: 44).    A linearized vector of 8740 bp was obtained and was gel purified.

The EPO insert was prepared by overlap PCR as follows. The first PCRproduct was produced by amplification of a synthetic EPO sequence clonedinto a standard cloning vector with Pfu polymerase and the followingprimers: 5′pNLB/Epo (5′-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3′) (SEQ ID NO:45) and pNLB/3′Epo (5′-TCCCCATACTAGACTTTTTACCTATCGCCGGTC-3′) (SEQ ID NO:46). The second PCR product was produced by amplification of a region ofpNLB-CMV-hIFN alpha-2b with Pfu polymerase and the following primers:3′Epo/pNLB (5′-ACCGGCGATAGGTAAAAAGTCTAGTATGGG-3′) (SEQ ID NO: 47) andpNLB/SapI (5′-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3′) (SEQ ID NO: 48).The two PCR products were mixed and reamplified with the followingprimers: 5′pNLB/Epo (5′-GGGGGGAAGCTTTCACCATGGGCGTGCACGAG-3′) (SEQ ID NO:45) and pNLB/SapI (5′-GGGGGGGCTCTTCTCAGCTGGAATTCCGCCGATAC-3′) (SEQ IDNO: 48).

The fusion PCR product was digested with Hind III and Eco RI and a 633bp fragment gel purified. The 8740 bp and 633 bp fragments were ligatedto create pNLB-CMV-EPO.

EPO 1 - Synthetic EPO sequence (610 nt) (SEQ ID NO: 42)AAGCTTTCACCATGGGCGTGCACGAGTGCCCTGCTTGGCTGTGGCTGCTCTTGAGCCTGCTCAGCCTGCCTCTGGGCCTGCCTGTGCTGGGCGCTCCTCCAAGGCTGATCTGCGATAGCAGGGTGCTGGAGAGGTACCTGCTGGAGGCTAAGGAGGCTGAGAACATCACCACCGGCTGCGCTGAGCACTGCAGCCTGAACGAGAACATCACCGTGCCTGATACCAAGGTGAACTTTTACGCTTGGAAGAGGATGGAGGTGGGCCAGCAGGCTGTGGAGGTGTGGCAGGGCCTGGCTCTGCTGAGCGAGGCTGTGCTGAGGGGCCAGGCTCTGCTGGTGAACAGCTCTCAGCCTTGGGAGCCTCTGCAGCTGCACGTGGATAAGGCTGTGAGCGGCCTGAGAAGCCTGACCACCCTGCTGAGGGCTCTGAGGGCTCAGAAGGAGGCTATCAGCCCTCCAGATGCTGCAAGCGCTGCCCCTCTGAGGACCATCACCGCTGATACCTTTAGGAAGCTGTTTAGGGTGTACAGCAACTTTCTGAGGGGCAAGCTGAAGCTGTACACCGGCGAGGCTTGCAGGACCGGCGATAGGTAAAAAGGCC GGCCGAGCTC

An EPO coding sequence is produced which codes for a 165 amino acid formof EPO with the terminal codon (coding for arginine at position 166)removed. A 179 bp region of pNLB-CMV-EPO corresponding to the sequencethat extends from an Eco 47III site that resides in the EPO codingsequence to an EcoRI site that resides downstream of the EPO stop codonin pNLB-CMV-EPO was synthesized with the terminal arginine codon(position 166) eliminated so that aspartic acid (amino acid 165) will bethe terminal amino acid codon, resulting in a 176 bp Eco 47III/EcoRIfragment. The fragment was synthesized by Integrated DNA Technologies(Coralville, Iowa 52241) and cloned into a pDRIVE vector (Qiagen, Inc),creating pDRIVE-des-Arg166-EPO. The 176 bp Eco 47III/EcoRI fragment wassubcloned into the Eco47III/EcoRI site of pNLB-CMV-EPO, creatingpNLB-CMV-Des-Arg166-EPO.

Transduction particles were prepared from the pNLB-CMV-Des-Arg166-EPOessentially as described in Example 2.

EXAMPLE 23 Production of Transgenic Chickens Expressing HumanErythopoietin

1234 White Leghorn chicken eggs were windowed and injected with thetransduction particles essentially as described in Example 2. 334 of theeggs hatched. DNA was extracted from rooster sperm samples by Chelex-100extraction (Walsh et al., 1991). DNA samples were then subjected toTaqman™ analysis on a 7700 Sequence Detector (Perkin Elmer) to detectthe transgene. Seven of the hatched G0 roosters tested positive for theNLB-CMV-EPO transgene. Three of the chimeric germline transgenicroosters that tested positive for the NLB-CMV-EPO transgene were bred tonon-transgenic females by artificial insemination to produce 1190offspring, 14 of which were transgene positive germline transgenic G1's.Egg white of eggs laid by the G1 germline transgenic females or theirdescendents contained about 0.4 to 1.9 μg/ml of EPO, as determined byELISA.

EXAMPLE 24 Purification of Transgenic Poultry Derived EPO

Egg white from eggs of transgenic chickens which produce EPO in theiroviduct was diluted with three volumes of 50 mM sodium acetate, pH 4.6,mixed and then filtered and loaded on to a Sepharose cation exchangecolumn. Following a wash of the column with 50 mM sodium acetate, pH5.0, containing 100 mM NaCl, the EPO was eluted with the same acetatebuffer containing 500 mM NaCl together with 0.05% Tween 20. The EPOeluted from the Sepharose column was loaded on to a Phenyl Sepharosehydrophobic interaction chromatography column. The column wasequilibrated with 2 M NaCl, 50 mM Tris-HCl, pH 7.2, 0.05% Tween 20. Thesame buffer was used to wash the column after loading of thepreparation. This is followed by a water wash. EPO was subsequentlyeluted with 30% IPA. The EPO preparation was then applied to areversed-phase HPLC column and the EPO eluted with an increasingconcentration of ethanol in 0.1% trifluoroacetic acid. The peak of EPOelution occurs at an ethanol concentration of about 53%. Diafiltrationwas used to concentrate the final EPO preparation and to replace thesolvent with 0.1 M sodium phosphate buffer, pH 7.0.

EXAMPLE 25 Carbohydrate Analysis of Transgenic Poultry DerivedErythropoietin

The oligosaccharide structures were determined for avian derived humanEPO by employing the following analysis techniques as are well known topractitioners of ordinary skill in the art.

The O-linked oligosaccharides were chemically released from the proteinand the N-linked oligosaccharides were enzymatically released from theprotein. After release, the O-linked and the N-linked oligosaccharideswere permethylated using the NaOH method involving reaction with methyliodide under anhydrous DMSO and were then extracted into chloroformprior to analysis. The structures were separated using HPLC.

MALDI-TOF-MS (Matrix assisted laser desorption ionization time-of-flightmass spectrometry) analysis and ESI MS/MS (electrospray ionizationtandem mass spectrometry) were performed on the oligosaccharides afterrelease from the peptide backbone and purification as is understood inthe art. Samples of the individual polysaccharide species were alsodigested with certain enzymes and the digest products were analyzed byHPLC as is understood in the art.

The O-linked and N-linked oligosaccharide structures shown below wereidentified. Linkage analysis of the structures revealed the linkagesshown in FIGS. 20 and 21.

N-linked EPO structures are shown below.

O-linked EPO structures are shown below.

wherein

-   Gal=Galactose,-   NAcGal=N-Acetyl-Galactosamine,-   NAcGlu=N-Acetyl-Glucosamine, and-   SA=Sialic Acid.

EXAMPLE 26 Carbohydrate Analysis of Transgenic Poultry Derived EPO

Monosaccharide analysis of EPO obtained from a transgenic chicken wasperformed by GC/MS (gas chromatography-mass spectrometry). The samplewas spiked with Arabitol (internal standard), hydrolyzed, N-acetylatedand TMS derivatized using methods readily available to those skilled inthe art. The derivatized sample was compared to a standard mixture ofsugars similarly derivatized. Sialic acid analysis of the EPO wasperformed after spiking with ketodeoxynonulosonic acid, lyophilizingthen hydrolyzing, desalting and re-lyophilizing. Analysis of the samplewas performed on a Dionex BioLC system using appropriate standards.Table 6 shows the quantification of monosaccharides detected for theEPO. Trace amounts of contaminating xylose, fucose and glucose were alsodetected in the monosaccharide analysis. The data in Table 6 supersedespreliminary data generated by HPAEC-PAD analysis.

TABLE 6 nmoles nmoles detected/mg Monosaccharide detected sample Mannose49 245 Galactose 16 80 N-Acetylgalactosamine 6.0 30 N-Acetylglucosamine91 455 Sialic acid 4.7 24

EXAMPLE 27 In Vitro Cell Proliferation Activity of TPD Human EPO

The in vitro biological activity of the poultry derived human EPO wasdemonstrated using the TF-1 cell proliferation assay. Two separatesamples representing two fractions (SP1 column: 130 mM NaCl and 250 mMNaCl) recovered from an initial ion exchange purification step weretested. Each of the two fractions showed essentially the same cellproliferation activity and it was also subsequently shown that theglycosylated erythropoietin contained in the two fractions wasessentially the same. Briefly, TF-1 cells were maintained in growthmedia containing GM-CSF (2 ng/ml). Confluent cultures were harvested,washed and plated in wells of a standard 96 well plate (each well 0.32cm²) at a cell density of 10⁴ cells per well in growth media notcontaining GM-CSF. Avian derived EPO was serial diluted in growth mediaand added to separate wells in triplicate. Cell proliferation after 5days was determined by metabolic reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) andwas quantified spectrophotometrically. The in vitro activity of thepurified EPO is shown in FIG. 22.

All documents (e.g., U.S. patents, U.S. patent applications,publications) cited in the above specification are incorporated hereinby reference. Various modifications and variations of the presentinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A composition comprising an interferon-α molecule glycosylated with:Gal-NAcGal-, wherein Gal=Galactose, and NAcGal=N-Acetyl-Galactosamine.2. The composition of claim 1 wherein the composition is apharmaceutical composition.
 3. The composition of claim 1 wherein theinterferon-α is interferon-α2.
 4. The composition of claim 1 wherein theinterferon-α is interferon-α2b.
 5. The composition of claim 1 whereinthe glycosylation is O-linked at Thr-106.
 6. The composition of claim 1wherein the interferon-α is human interferon-α.
 7. The composition ofclaim 1 wherein the interferon-α is pegylated.
 8. A compositioncomprising an interferon-α molecule glycosylated with:Gal-Gal-NAcGal-, wherein Gal=Galactose, andNAcGal=N-Acetyl-Galactosamine, wherein the glycosylation is O-linked atThr-106.
 9. A composition comprising an interferon-α moleculeglycosylated with:Gal-Gal-NAcGal-, wherein Gal=Galactose, andNAcGal=N-Acetyl-Galactosamine, wherein the interferon-α is humaninterferon-α.
 10. A composition comprising an interferon-α moleculeglycosylated with:Gal-Gal-NAcGal-, wherein Gal=Galactose, andNAcGal=N-Acetyl-Galactosamine, wherein the interferon-α is pegylated.